Protein screening methods

ABSTRACT

The invention provides methods and compositions useful for identifying polypeptides with desired characteristics in vitro.

RELATED APPLICATIONS

This application is a continuation of U.S. Utility application Ser. No.13/009,500, filed Jan. 19, 2011 which is a continuation application ofPCT/US2009/051716, filed Jul. 24, 2009, and which claims priority toU.S. Provisional Application No. 61/083,813, filed Jul. 25, 2008, U.S.Provisional Application No. 61/090,111, filed Aug. 19, 2008, and U.S.Provisional Application No. 61/170,029, filed Apr. 16, 2009, thecontents of which are hereby incorporated by reference.

BACKGROUND

It is well understood that, in the use of display and selectiontechnologies, access to greater diversity allows for a more effectiveselection of molecules with the highest affinity, specificity,stability, and/or other desirable characteristics.

Past methods that have been developed include phage display, ribosomedisplay, CIS display, and mRNA display. Recently, there has beenincreased interest in identifying molecules using in vivo screening (JControl Release. 2003 Aug. 28; 91(1-2):183-6). This approach has beenmade possible using phage display, but suffers from the limiteddiversity afforded by the phage display technologies. Ribosome or mRNAdisplay would fail for this application due to the instability of theRNA species. Therefore, development of DNA-protein fusions would behighly desirable, due to the increased stability of the species.

Three types of DNA-protein fusions have been described. CIS display isone method where coupled in vitro transcription/translation is used togenerate a dsDNA binding protein that covalently binds to the DNA as itis being transcribed and translated (PNAS, 101(9): 2806-2810). However,one of the primary limitations of this technology is that thesynthesized protein can bind to any neighboring DNAs that are nearbyduring the transcription/translation process, resulting in mis-taggedfusions. The second method is that of Kurz and Lohse (Chembiochem. 2001Sep. 3; 2(9):666-72). This method involves formation of covalent adductswith mRNA using a multifunctional species that can covalently bond withtranslated protein, create a ribosomal pause at the covalent adduct onRNA, and serve as a primer for reverse transcription. A limitation ofthis method is the inefficiency of the covalent linking step with RNAusing psoralen. The third method is that of Yonezawa et al. (NucleicAcids Res. 2003 Oct. 1; 31(19): e118.) In this method, DNA encodingstreptavidin and a region of diverse peptides is biotinylated, placed ina synthetic microsphere with translation machinery and translated suchthat the streptavidin (tetrameric) will bind to the biotinylated DNA.The limitation of this method is that the resultant species istetrameric, which can be troublesome for affinity selections due tomultiple binding species on one particle (rebinding effect).

Herein is described a simple, efficient method for generating nucleicacid protein fusions is required which may employ noncovalent attachmentbetween the nucleic acid and the protein, and which will allow theformation of DNA-protein fusions.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods to select andevolve desired properties of proteins and nucleic acids. In variousembodiments, the current invention includes small molecules linked tonucleic acid or modified nucleic acid. Other embodiments include methodsfor producing polypeptides, including peptides with modified aminoacids, assays for enabling selection of individual members of thepopulation having desired properties, and methods for generating newvariations of polypeptides with improved properties.

In one aspect the invention provides a heterobifunctional complexcomprising:

-   -   (a) a first nucleic acid molecule comprising a        polypeptide-encoding sequence;    -   (b) a polypeptide encoded by the first nucleic acid molecule;        and    -   (c) a second nucleic acid molecule comprising a nucleic sequence    -   complementary to a portion of the first nucleic acid molecule,        wherein the first nucleic acid molecule is bound to the second        nucleic molecule through complementary nucleic acid base        pairing, and wherein the second nucleic acid molecule is        non-covalently bound to the polypeptide.

In some embodiments, this heterobifunctional complex further comprises:

-   -   (a) a high affinity ligand covalently bound to the second        nucleic acid molecule; and    -   (b) a ligand acceptor bound to a peptide acceptor,        wherein the high affinity ligand is bound to the ligand acceptor        and the peptide acceptor is covalently bound to the polypeptide.        In some embodiments, this heterobifunctional complex further        comprises a second high affinity ligand, wherein the second high        affinity ligand is covalently bound to the peptide acceptor, and        wherein the second high affinity ligand is bound to the ligand        acceptor. In some embodiments, the peptide acceptor in this        heterobifunctional complex is bound to the second high affinity        ligand through a linker. In one preferred embodiment, the linker        comprises polyethylene glyocol. In another preferred embodiment,        the linker further comprises a polysialic acid linker. In some        embodiments, the ligand acceptor in this heterobifunctional        complex is covalently bound to the peptide acceptor.

In some embodiments, this heterobifunctional complex further comprises:

-   -   (a) a ligand acceptor covalently bound to the second nucleic        acid molecule; and,    -   (b) a high affinity ligand bound to a peptide acceptor, wherein        the ligand acceptor is bound to the high affinity ligand and the        peptide acceptor is covalently bound to the C-terminus of the        polypeptide.

In other embodiments, this heterobifunctional complex further comprisesa third nucleic acid molecule, comprising a nucleic sequencecomplementary to a portion of the second nucleic acid molecule, whereinthe third nucleic acid molecule is bound to the second nucleic moleculethrough complementary nucleic acid base pairing, wherein the thirdnucleic acid molecule is covalently bound to a peptide acceptor, andwherein the peptide acceptor is covalently bound to the polypeptide.

In some embodiments, the second nucleic acid molecule in the complexesdescribed above is a branched nucleic acid molecule. In someembodiments, the second nucleic acid molecule in the complexes describedabove is capable of acting as a primer for reverse transcription of thefirst nucleic acid molecule.

In another aspect, this invention provides an X-display complex (e.g., anucleic acid polypeptide complex) comprising:

-   -   (a) a nucleic acid molecule comprising a polypeptide-encoding        sequence, covalently bound to a first high affinity ligand;    -   (b) a polypeptide encoded by the nucleic acid molecule;    -   (c) a ligand acceptor bound to a peptide acceptor, wherein the        high affinity ligand is bound to the ligand acceptor and the        peptide acceptor is covalently bound to the C-terminus of the        polypeptide.

In some embodiments, the ligand acceptor in this X-display complex iscovalently bound to the peptide acceptor. In some embodiments, thisX-display complex further comprises a second high affinity ligand,wherein the second high affinity ligand is covalently bound to thepeptide acceptor, and wherein the second high affinity ligand is boundto the ligand acceptor.

In another aspect, this invention provides an X-display complexcomprising:

-   -   (a) a nucleic acid molecule comprising a polypeptide-encoding        sequence, covalently bound to a first high affinity ligand;    -   (b) a first ligand acceptor covalently bound to a second high        affinity ligand; and,    -   (c) a polypeptide encoded by the first nucleic acid molecule,        wherein the polypeptide comprises a second ligand acceptor,    -   wherein the first high affinity ligand is bound to the first        ligand acceptor, and the second high affinity ligand is bound to        the second ligand acceptor.

In some embodiments, the first or second high affinity ligand bound tothe complexes described above is biotin.

In some embodiments, the first or second high affinity ligand bound tothe complexes described above is selected from the group comprisingFK506, methotrexate, PPI-2458, biotin, hirudin, ZFVp(O)F,gluorescein-biotin, ABD (albumin binding domain), 18 bp DNA, RNAse A,cloroalkanes, Aryl (beta-amino ethyl) ketones, and Protein A.

In some embodiments, the first or second ligand acceptor in thecomplexes described above is selected from the group comprising FKBP12,dihydrofolate reductase, methionine aminopeptidase, dimericstreptavidin, streptavidin tetramer, thrombin, carboxypeptidase,Monovalent Ab, HSA (albumin), Zn finger, hRI (RNase inhibitor), mutatedhaloalkane dehalogenase, haloTag, and sortase.

In some embodiments, the first or second ligand acceptor in thecomplexes described above is streptavidin.

In some embodiments, the polypeptide described above is chosen from thegroup comprising an antibody, a VH domain, a VL domain, a Fab fragment,a single chain antibody, a nanobody, a unibody, an adnectin, anaffibody, a DARPin, an anticalin, an avimer, a ¹⁰Fn3 domain, and aversabody.

In another aspect, this invention provides an X-display complexcomprising:

-   -   (a) a nucleic acid molecule comprising a polypeptide-encoding        sequence, covalently attached to a ligand; and    -   (b) a polypeptide encoded by the first nucleic acid molecule,        wherein the polypeptide comprises a ligand acceptor,        wherein the ligand is bound to the ligand acceptor.

In some embodiments, the ligand bound to the X-display complex is FK506and the ligand acceptor in the nucleic acid-polypeptide complexes is theFK506-binding domain of FKBP.

In some embodiments, the first nucleic acid molecule bound to thecomplexes described above is selected from the group consisting ofssRNA, ssDNA, ssDNA/RNA hybrid dsDNA, and dsDNA/RNA hybrid.

In some embodiments, the polypeptide-encoding sequence of the firstnucleic acid molecule bound to the complexes described above does notcontain an in-frame stop codon.

In some embodiments, the polypeptide described above is a bindingprotein. In one preferred embodiment, the binding protein is the VH orVL domain of an antibody.

In some embodiments, the nucleic acid-polypeptide complexes describedabove does not contain a ribosome.

In another aspect, this invention also provides a library comprising aplurality of the X-display complexes described above, wherein at least aportion of the complexes contain different polypeptide-encodingsequences.

In another aspect, this invention also provides a method of a library ofnucleic acid-polypeptide complexes comprising the steps of:

-   -   providing a library of mRNA sequences comprising a sequence        element complementary to a first nucleic acid linker    -   providing a first nucleic acid linker operably linked to a first        high affinity ligand such that the first nucleic acid linker        binds to the mRNA through complementary nucleic acid base        pairing    -   providing second high affinity ligand operably linked to a        peptide acceptor    -   providing a ligand acceptor with at least two binding sites or        providing at least such that the ligand acceptor binds to the        first high affinity ligand and the second high affinity ligand    -   allowing translation of the mRNA to occur such that the peptide        acceptor binds to the translated protein thereby forming a        nucleic acid-polypeptide complex linking the mRNA to the        protein.

In some embodiments, the method further comprises

-   -   allowing reverse transcription of the mRNA using the first        nucleic acid linker in the X-display complex as a primer such        that a DNA/RNA hybrid is formed.

In one preferred embodiment, the method further comprises

-   -   degrading the mRNA and synthesizing a complementary DNA strand        thereby forming a DNA/DNA hybrid in the nucleic acid-polypeptide        complex.

In some embodiments, the mRNA in the library further comprises a TMVenhancer.

In some embodiments, the mRNA in the library further comprises a Cμsequence.

In some embodiments, the mRNA in the library further comprises a FLAGtag.

In some embodiments, the mRNA in the library further comprises an SAdisplay sequence.

In some embodiments, the mRNA in the library further comprises an A20tail.

In another aspect, this invention also provides a library of nucleicacid-polypeptide complexes produced by the methods described in thisinvention.

In another aspect, this invention also provides a method of selecting anisolated nucleic acid molecule encoding a polypeptide capable of bindingto an antigen of interest, comprising the steps of:

-   -   (a) providing the library of X-display complexes described in        this invention;    -   (b) contacting the library with an antigen of interest;    -   (c) selecting from the library at least one X-display complex        that binds to the antigen of interest; and,    -   (d) isolating the polypeptide encoding sequence of the selected        X-display complex.

In another aspect, this invention also provides a method of producing apolypeptide capable of binding to an antigen of interest, comprisingintroducing a polypeptide encoding sequence identified by the methoddescribed in this invention into an expression environment such that theencoded polypeptide is produced.

In another aspect, this invention also provides an isolated nucleic acidmolecule encoding a polypeptide capable of binding to an antigen ofinterest, selected by the method described in this invention.

In another aspect, this invention also provides a X-display complexcomprising:

(a) a first nucleic acid molecule comprising a polypeptide-encodingsequence;

(b) a polypeptide encoded by the first nucleic acid molecule;

(c) a second nucleic acid molecule comprising a nucleic sequencecomplementary to a portion of the first nucleic acid molecule;

(d) a first high affinity ligand covalently bound to the second nucleicacid molecule;

(e) a first ligand acceptor; and

(f) a second high affinity ligand covalently bound through via one ormore linking molecules to a peptide acceptor,

wherein the first nucleic acid molecule is bound to the second nucleicmolecule through complementary nucleic acid base pairing,wherein the first high affinity ligand is noncovalently bound to theligand acceptor at a first binding site,wherein the second ligand is noncovalently bound to the ligand acceptorat a second binding site, andwherein the one or more linking molecules are polyethylene glycolmolecules.

In some embodiments, the first high affinity ligand in this complex isbiotin and the ligand acceptor is selected from the group comprisingstreptavidin, dimeric streptavidin, and tetrameric streptavidin.

In some embodiments, the second high affinity ligand in this complex isbiotin and the ligand acceptor is selected from the group comprisingstreptavidin, dimeric streptavidin, and tetrameric streptavidin.

In some embodiments, the first or second high affinity ligand in thiscomplex is selected from the group comprising FK506, methotrexate,PPI-2458, biotin, hirudin, ZFVp(O)F, fluorescein-biotin, ABD (albuminbinding domain), 18 bp DNA, RNAse A, cloroalkanes, aryl (beta-aminoethyl) ketones, and protein A.

In some embodiments, this complex further comprises a second ligandacceptor.

In some embodiments, the second ligand acceptor in the complex describedabove is selected from the group comprising FKBP12, dihydrofolatereductase, methionine aminopeptidase, dimeric streptavidin, streptavidintetramer, thrombin, carboxypeptidase, monovalent Ab, HSA (albumin), Znfinger, hRI (RNase inhibitor), mutated haloalkane dehalogenase, haloTag,and sortase.

In some embodiments, the peptide acceptor in this complex is puromycin.

In some embodiments, the first or second nucleic acid molecule in thiscomplex further comprises psoralen.

In some embodiments, the polypeptide is chosen from the group comprisingan antibody, a VH domain, a VL domain, a Fab fragment, a single chainantibody, a nanobody, a unibody, an adnectin, an affibody, a DARPin, ananticalin, an avimer, a ¹⁰Fn3 domain, and a versabody.

In another aspect, this invention provides a heterobifunctional complexcomprising:

(a) a first high affinity ligand covalently bound to a nucleic acidmolecule;

(b) a second high affinity ligand covalently bound to a peptideacceptor; and

(c) a ligand acceptor comprising two or more ligand binding sites;

wherein the first and second are bound to the ligand acceptor atdistinct ligand binding sites.

In some embodiments, the first and the second high affinity ligand inthis complex are identical.

In some embodiments, the first and the second high affinity ligand inthis complex are biotin.

In some embodiments, the nucleic acid molecule in this complex comprisesa psoralen moiety.

In some embodiments, the peptide acceptor in this complex is puromycin.

In some embodiments, the ligand acceptor in this complex is a multimericprotein.

In some embodiments, the ligand acceptor in this complex isstreptavidin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates one embodiment of the methodology described herein.

FIG. 2 illustrates the same using an advanced primer that is an invertedpolarity primer with two 3′ ends that can conveniently prime a cDNA andcarry a puromycin or derivative or small molecule on the opposite end

FIG. 3 illustrates the same using an advanced primer that is a stem loopvariety and can double back and conveniently prime a cDNA.

FIG. 4 illustrates one embodiment of the methodology being employed toimprove or evolve the structure/function of an antibody heavy chainvariable region wherein the polypeptide is fused to an FK12BP proteinthat can non covalently bind to the nucleic acid linked FK506 smallmolecule thereby linking an improved (evolved) phenotype (polypeptide)with its encoding genotype (nucleic acid).

FIG. 5 illustrates one embodiment of the methodology of in vitro displayusing a branched oligonucleotide linker and a complementaryoligonucleotide comprising a covalently attached peptide acceptor

FIG. 6 illustrates one embodiment of the methodology of in vitro displayusing a branched oligonucleotide linker, covalently attached to apeptide acceptor

FIG. 7 illustrates one embodiment of the methodology of in vitro displayusing high affinity ligands and ligand acceptors.

FIG. 8 illustrates one embodiment of the methodology of in vitro displayusing high affinity ligands and ligand acceptors, wherein the ligandacceptor is covalently linked to a peptide acceptor.

FIG. 9 illustrates one embodiment of the methodology of in vitro displayusing high affinity ligands and ligand acceptors, wherein the ligandacceptor is non-covalently linked to a peptide acceptor.

FIG. 10 illustrates one embodiment of the methodology of in vitrodisplay using high affinity ligands and ligand acceptors, wherein afirst ligand acceptor molecule is covalently linked to a second(non-cognate) high affinity ligand, wherein the first ligand acceptormolecule can bind to a cognate first high affinity ligand covalentlylinked to a nucleic acid, and wherein the second high affinity ligandcan bind to a cognate second ligand acceptor fused to the polypeptideencoded by the nucleic acid.

FIG. 11 illustrates a non-limiting example of the individual componentsused in one embodiment of the methodology of in vitro display using highaffinity ligands and ligand acceptors.

FIG. 12 illustrates a non-limiting assembly order of nucleic/proteincomplexes for one embodiment of the methodology of in vitro displayusing high affinity ligands and ligand acceptors.

FIG. 13 illustrates the design of a preferred X-display complex.

FIG. 14 illustrates an exemplary scheme for preparing a VH library,assembling the display complex, selecting, and purifying the displaycomplex.

FIG. 15 (SEQ ID NOS: 66, 52, 54, and 67) illustrates a sample singleclone for a VH library. The illustration shows transcription andtranslation start sequences, VH sequence, Cu sequence, FLAG tagsequence, a DNA segment complementary to a linker (e.g., an NA linker)

FIGS. 16A-16B illustrate the steps of a preferred embodiment of thedisplay methodology. FIG. 16A first depicts an X-display complexcomprising an mRNA molecule (containing appropriate linkers, tags,complementary regions, and a poly-A tail) which is attached to aLinker/RTprimer (an NA linker) via complementary strand pairing and apsoralen link. The NA linker is further covalently bound to a Biotin (B)molecule which is associated with a streptavidin molecule. Thestreptavidin is associated with a second biotin molecule, which itselfis covalently bound to puromycin which has been attached to a translatedprotein. FIG. 16A (SEQ ID NOS: 68 and 69) depicts the reversetranscription (Step 1) and RNA degradation (Step 2). FIG. 16B depictssecond strand synthesis to create a DNA/RNA hybrid (Step 4). The finaldepiction of FIG. 16B is the final dsDNA X-display complex (i.e.,X-display complex).

FIG. 17 (SEQ ID NOS: 61, 62, 70, and 71) depicts a potential librarymember and how, in some embodiments, primers may be employed with 454sequencing.

FIG. 18 (SEQ ID NOS: 72-78) depicts an exemplary segment of a VH librarymember.

FIG. 19 depicts an exemplary segment of a display library member andfurther displays end sequences from the primers (see FIG. 17).

FIG. 20 (SEQ ID NOS: 72-74 and 79-81) depicts an exemplary segment of adisplay library member and further provides the exemplary sequence theTMV UTR and C-mu regions from the primers.

FIG. 21 (SEQ ID NOS: 59, 82-89) depicts a possible arrangement ofprimers for amplifying a clone selected for sequencing.

FIG. 22 (SEQ ID NOS: 68, 90 and 91) illustrates the progress of oneembodiment of the invention from library through selection, illustratingthat the method may be iterative such that nucleic acids/proteinsselected in one round may be used to generate a library for subsequentrounds of X-display complex formation and selection.

FIG. 23 (SEQ ID NO: 68) illustrates one embodiment of the inventionwhereby several rounds of selection are employed without regenerating alibrary or amplifying the selection products.

FIG. 24 illustrates one method of VH and VL library design from mRNA.

FIG. 25 illustrates one exemplary embodiment of selection method whereinthe target molecule used in affinity selection is immobilized on a solidsupport.

FIG. 26 illustrates one exemplary embodiment of selection method whereinthe target molecule used in affinity selection is expressed on a cellsurface. In such an embodiment the display complex is selected againstcells not expressing the complex (e.g., in order to remove complexeswhich bind the cells but do not bind the target of interest) and thenselected against cells which express the target of interest (i.e., toidentify specific binders).

FIG. 27 illustrates a DNA X-display complex (i.e., a DNA-protein fusion)wherein source tags have been incorporated into the nucleic acid (e.g.,to identify the source of the encoding DNA) and a constant sourceencoding used to tag nucleic acids from different rounds of selections(useful, e.g., when pooling nucleic acid from different selection roundsfor one sequencing run).

FIG. 28 illustrates a DNA X-display complex (i.e., a DNA-protein fusion)and the addition of an N6 primer.

FIG. 29 depicts a flow chart showing one embodiment of a sequenceanalysis scheme.

FIG. 30 illustrates an exemplary result of mRNA transcription,purification and crosslinking process.

FIG. 31 illustrates an exemplary result of mRNA transcription,purification and streptavidine loading process.

FIG. 32 illustrates an exemplary result after RNaseH treatment andelution process.

FIG. 33 illustrates an exemplary result after RNaseH treatment andelution process.

FIG. 34 illustrates an exemplary result after RT-PCR and 2^(nd) strandsynthesis process.

FIG. 35 (SEQ ID NOS: 82-89, 59) illustrates the pool tagging which maybe used in the library design to allow for pooling of multiple selectionrounds into one sequencing run.

FIG. 36 illustrates exemplary donor cells for the preparation of a VHlibrary.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compositions and methods to select andevolve desired properties of proteins and nucleic acids. The method maybe referred to herein as “X-Display” or, in some embodiments werestreptavidin is employed, “SA Display.” In various embodiments, thecurrent invention includes small molecules linked to nucleic acid ormodified nucleic acid. Other embodiments include methods for producingpolypeptides, including peptides with modified amino acids, assays forenabling selection of individual members of the population havingdesired properties, and methods for generating new variations ofpolypeptides with improved properties.

By “selecting” is meant substantially partitioning a molecule from othermolecules in a population. As used herein, a “selecting” step providesat least a 2-fold, in some embodiments 3-fold, 5-fold, 10-fold, 20-fold,preferably 30-fold, more preferably, a 100-fold, and, most preferably, a1000-fold enrichment of a desired molecule relative to undesiredmolecules in a population following the selection step. A selection stepmay be repeated any number of times, and different types of selectionsteps may be combined in a given approach.

By a “protein” is meant any two or more naturally occurring or modifiedamino acids joined by one or more peptide bonds. “Protein” and “peptide”are used interchangeably herein.

By “RNA” is meant a sequence of two or more covalently bonded, naturallyoccurring or modified ribonucleotides. One example of a modified RNAincluded within this term is phosphorothioate RNA.

By “DNA” is meant a sequence of two or more covalently bonded, naturallyoccurring or modified deoxyribonucleotides.

By a “nucleic acid” is meant any two or more covalently bondednucleotides or nucleotide analogs or derivatives. As used herein, thisterm includes, without limitation, DNA, RNA, and PNA. The term “nucleicacid” may include a modified nucleic acid, and, accordingly, nucleicacid and modified nucleic acid may be used interchangeably.

The term “nucleotide analog” or “nucleotide derivative” or “modifiednucleic acid” as used herein refers to modified or non-naturallyoccurring nucleotides such as 5-propynyl pyrimidines (i.e.,5-propynyl-dTTP and 5-propynyl-dTCP), 7-deaza purines (i.e.,7-deaza-dATP and 7-deaza-dGTP). Nucleotide analogs include base analogsand comprise modified forms of deoxyribonucleotides as well asribonucleotides.

By a “peptide acceptor” is meant any molecule capable of being added tothe C-terminus of a growing protein chain by the catalytic activity ofthe ribosomal peptidyl transferase function. Typically, such moleculescontain (i) a nucleotide or nucleotide-like moiety (for example,adenosine or an adenosine analog (di-methylation at the N-6 aminoposition is acceptable)), (ii) an amino acid or amino acid-like moiety(for example, any of the 20 D- or L-amino acids or any amino acid analogthereof (for example, O-methyl tyrosine or any of the analogs describedby Ellman et al., Meth. Enzymol. 202:301, 1991), and (iii) a linkagebetween the two (for example, an ester, amide, or ketone linkage at the3′ position or, less preferably, the 2′ position); preferably, thislinkage does not significantly perturb the pucker of the ring from thenatural ribonucleotide conformation. Peptide acceptors may also possessa nucleophile, which may be, without limitation, an amino group, ahydroxyl group, or a sulfhydryl group. In addition, peptide acceptorsmay be composed of nucleotide mimetics, amino acid mimetics, or mimeticsof the combined nucleotide-amino acid structure.

As used herein, the terms “X-display complex,” “nucleic acid-polypeptidecomplex,” and “nucleic acid-protein fusions” are interchangeable and aremeant to refer to a complex formed from the interaction, either directlyor indirectly, of a nucleic acid and a protein (or peptide or fragmentthereof) encoded by the nucleic acid. In some instances the X-displaycomplexes may be referred to “display complexes” or “display fusions”and the skilled artisan will understand by the context of such use thatit should not be confused with other types of display systems which maybe referenced herein. In some embodiments the nucleic acid is RNA, e.g.,mRNA. In other preferred embodiments the nucleic acid is DNA or cDNA.Accordingly, in some embodiments where the goal is to display a complexcontaining DNA, the X-display complex may be called a “DNA-proteinfusion” or “DNA-display fusion.” It is important to note that the use ofthe term “fusion” does not imply or suggest that the nucleic acid iscovalently attached to the peptide or protein. In some embodiments theRNA or DNA may be modified or altered. In preferred embodiments theinteraction between the nucleic acid and the protein is noncovalent(e.g., the interaction may be mediated by biotin/streptavidininteractions, linker molecules, and the like as described herein). Inpreferred embodiments the nucleic acid is mRNA or modified mRNA.

By an “altered function” is meant any qualitative or quantitative changein the function of a molecule.

By “binding partner,” as used herein, is meant any molecule which has aspecific, covalent or non-covalent affinity for a portion of a desiredDNA-protein fusion. Examples of binding partners include, withoutlimitation, members of antigen/antibody pairs, protein/inhibitor pairs,receptor/ligand pairs (for example cell surface receptor/ligand pairs,such as hormone receptor/peptide hormone pairs), enzyme/substrate pairs(for example, kinase/substrate pairs), lectin/carbohydrate pairs,oligomeric or heterooligomeric protein aggregates, DNA bindingprotein/DNA binding site pairs, RNA/protein pairs, and nucleic acidduplexes, heteroduplexes, or ligated strands, as well as any moleculewhich is capable of forming one or more covalent or non-covalent bonds(for example, disulfide bonds) with any portion of a X-Display complex.

By a “solid support” is meant, without limitation, any column (or columnmaterial), bead, test tube, microtiter dish, solid particle (forexample, agarose or sepharose), microchip (for example, silicon,silicon-glass, or gold chip), or membrane (for example, the membrane ofa liposome or vesicle) to which an affinity complex may be bound, eitherdirectly or indirectly (for example, through other binding partnerintermediates such as other antibodies or Protein A), or in which anaffinity complex may be embedded (for example, through a receptor orchannel).

Nucleic Acid or Modified Nucleic Acid Linkers to Mediate the X-DisplayComplex

In one aspect of the invention a nucleic acid (e.g., a native mRNA ormodified mRNA) may be attached to its encoded protein at the end oftranslation by the use of a nucleic acid or modified nucleic acid linker(“NA linker”) which is hybridized at the 3′ end of the nucleic acid. Insuch an embodiment the NA linker has inverted polarity in the linker sothat it effectively has two 3′ ends, of which the non-hybridized end hasattached to it a puromycin or related analogue or a small moleculecapable of binding covalently or noncovalently with high affinity to thepolypeptide protein. Accordingly, in some embodiments, the X-displaycomplex is formed by the interaction of the protein and the nucleic acidwith the NA linker.

In some embodiments the NA linker is a Psoralen linker. In somepreferred embodiments the linker is XB-PBI. In some embodiments XB-DDBmay be used. In some preferred embodiments the linker (e.g., a PBIlinker or DDB linker) is attached to a high affinity ligand (e.g.,biotin), i.e., the linker includes the high affinity ligand.

In some embodiments a nucleic acid (e.g., a native mRNA or modifiedmRNA) may be cross linked to an NA linker which is further attached to apeptide acceptor or a high affinity ligand. Such cross-linking may beaccomplished by any methods known in the art. For example, U.S. Pat. No.6,416,950, which is incorporated herein by reference in its entirety)describes methods making such crosslinks (see, e.g., FIGS. 9-13 of U.S.Pat. No. 6,416,950).

In a further embodiment, the invention includes a dual function NAlinker with inverted 5′-3′ polarity in the linker, such that it iscapable of hybridizing to the nucleic acid, e.g. an mRNA or modifiedmRNA template, wherein the hybridization occurs on one 3′ end of themRNA outside of the coding region, and wherein the nucleic acid/modifiedlinker carries on its other 3′ end a peptide acceptor such as puromycinor functional analogues thereof (for example, but not limited to,pyrazolopyrimidine) or a small molecule capable of binding covalently orwith high affinity to the polypeptide protein.

In several embodiments, a nucleic acid, preferably an mRNA or modifiedmRNA, is hybridized to the NA linker at the 3′ end, which is itselfbound covalently or with high affinity to the polypeptide (or modifiedpolypeptide) through a covalent bond or by a high affinity noncovalentbond.

In further embodiments, the NA linker is capable of serving as a primerto reverse transcribe the mRNA.

An additional embodiment comprises an encoding nucleic acid operablylinked to a NA linker that carries a reverse polarity nucleic acidportion at its “5′ end” (in quotes, as it effectively has a 3′ polarityto direct polymerization), such that it can serve as a primer forpolymerization on the encoding nucleic acid template, in addition to itsability to bind, via a stem loop structure, to an NA linker that carriesa puromycin or related analogue or small molecule on its 3′ end (seeFIGS. 2 and 3).

In some embodiments, the encoding nucleic acid is operably linked to abranched NA linker, wherein a portion of the NA liner is complementaryto the 3′ end of the encoding nucleic acid (see FIG. 5). Any artrecognized means of generating a branched NA linker are contemplated.The branch point can occur at any location within the NA linker. Suchbranched NA linkers can also serve as primers for reverse transcriptionof the encoding nucleic acid, e.g., an mRNA, to which they are bound.

In some embodiments, the encoding nucleic acid is operably linked to abranched NA linker, wherein the linker is covalently attached to apeptide acceptor (see FIG. 6).

In some embodiments, the NA linker comprises locked nucleic acids (LNA)e.g., bicyclic nucleic acids where a ribonucleoside is linked betweenthe 2′-oxygen and the 4′-carbon atoms with a methylene unit. In aparticular embodiment, the region of the NA linker that is complementaryto the encoding nucleic acid comprises LNA, at least in part, toincrease nucleic acid duplex stability (see Kaur et al. (2006).Biochemistry 45 (23): 7347-55.

Ligand/Acceptor Linkage

In another aspect, the X-display complex (e.g., the encoding nucleicacid and the encoded polypeptide) is formed through the high affinity orcovalent binding of a high affinity ligand to its cognate bindingpartner (ligand acceptor). In such embodiments, a nucleic acid may belinked covalently or noncovalently to a high affinity ligand, whichin-turn binds to a ligand acceptor noncovalently or covalently. Inpreferred embodiments the interaction is noncovalent. In someembodiments the ligand acceptor is further associated with a second highaffinity ligand, which in turn is linked to a peptide acceptor.

Non-limiting examples of high affinity ligand/ligand acceptor pairsinclude, but are not limited to, FK506/FKBP12,methotrexate/dihydrofolate reductase, and PPI-2458/methionineaminopeptidase 2. Additional non-limiting examples of ligand/ligandacceptor pairs are shown in Table 1. In some embodiments, theligand/ligand acceptor pair is biotin/streptavidin. Any form ofstreptavidin is considered for use in the methods of the inventionincluding, but not limited to, monomeric strepavidin, dimericstrepavidin, tetrameric strepavidin, and chemically or geneticallymodified variants thereof. In some embodiments, the ligand acceptor istetrameric strepavidin. In a particular embodiment, the tetramericstrepavidin is chemically cross-linked to increase stability.

TABLE 1 Non-limiting examples of high affinity ligand/ligand acceptorpairs High Affinity Ligand Acceptor Ligand size Ligand Acceptor SizeK_(d) or K_(i) Reference Biotin Small Streptavidin 53 kDa 1-20 fMmolecule tetramer Hirudin 65 aa Thrombin 36 kDa 20 fM peptideZFV^(p)(O)F tripeptide carboxypeptidase 10-27 fM Biochemistry 1991, 30:8165-70 Fluorescein- Monovalent Ab 48 fM PNAS 2000, biotin 97: 10701-5ABD 46 aa HSA (albumin) 50-500 fM Prot. Eng. Des. (albumin Select. 2008,binding 21: 515-27 domain) 18 bp DNA Zn finger 6 Zn 2 fM PNAS 1998,finger 95: 2812-17 domains RNAse A 13 kDa hRI (RNase 50 kDa 290 aM-1 fMJMB 2007, inhibitor) 368: 434-449 Cloroalkanes Mutated Promegairreversible ACS haloalkane Chem. Bio dehalogenase, 2008, 3: 373-82HaloTag Inhibitors Small Sortase irreversible JBC 2007, Aryl (beta-molecules 282: 23129 amino ethyl) Identification ketones of sortase geneUSP7101692 Protein A Antibody 1 fM Fc domain

In some embodiments, the high affinity ligand is covalently linked tothe nucleic acid (ligand/nucleic acid molecule). Any art recognizedmethod of linking the high affinity ligand to the nucleic acid iscontemplated. In one embodiment, the high affinity ligand is covalentlylinked to 3′ end of an mRNA molecule.

In some embodiments, the high affinity ligand acceptor molecule iscovalently linked to a peptide acceptor, which in turn, can becomecovalently linked to a translated protein by the peptidyl transferaseactivity of a ribosome (see FIG. 8). The linkage of the high affinityligand acceptor to the peptide acceptor can be direct or via a linkermolecule. Any art recognized linker molecules are contemplated for usein the methods of the invention. In one embodiment, the high affinityligand acceptor is linked to the peptide acceptor using a polyethyleneglycol linker molecule.

In some embodiments, the high affinity ligand is covalently linked to apeptide acceptor, which in turn, can become covalently linked to atranslated protein by the peptidyl transferase activity of a ribosome(see FIG. 9). In some preferred embodiments, such ligand/peptideacceptor molecules can be non-covalently linked to a ligand/nucleic acidmolecule through a multimeric ligand acceptor, e.g., tetramericstrepavidin. The covalent linkage of the high affinity ligand to thepeptide acceptor can be direct or via a linker molecule. Any artrecognized linkers are contemplated for use in the methods of theinvention. In a particular embodiment, the high affinity ligand islinked to the peptide acceptor using a polyethylene glycol linkermolecule.

In some embodiments, the ligand acceptor molecule is fused to thepolypeptide encoded by the nucleic acid (see FIGS. 4 and 10). Suchfusion can be performed chemically, using chemical crosslinkers orgenetically. The ligand acceptor molecule can be fused to the encodedpolypeptide at any region. In a particular embodiment, the ligandacceptor molecule is genetically fused to the N terminal region of theencoded polypeptide.

In some embodiments, a first ligand acceptor molecule is covalentlylinked to a second (non-cognate) high affinity ligand. In suchembodiments the first ligand acceptor molecule may bind to a cognatefirst high affinity ligand which is covalently linked to a nucleic acid.The second high affinity ligand may bind to a cognate second ligandacceptor which is fused to the polypeptide encoded by the nucleic acid.

In a preferred embodiment, a ligand acceptor molecule, preferably amultivalent ligand acceptor (e.g., multivalent streptavidin), bindsnoncovalently to a first high affinity ligand (e.g., biotin), which iscovalently linked to a nucleic acid that is complementary to an mRNAmolecule. The multivalent ligand acceptor also binds noncovalently to asecond high affinity ligand (e.g., biotin), which is covalently linkedto peptide acceptor (e.g., puromycin). The second high affinity ligandmay be connected to the peptide acceptor directly or via a linker (asdescribed below, e.g., polyethylene glycol). In preferred embodimentsthe nucleic acid attached to the first high affinity ligand iscomplementary to the 3′ end of the mRNA. The nucleic acid (e.g., anucleic acid in an NA linker) should be at least long enough to stablybind to the protein encoding nucleic acid (e.g., mRNA) of the X-displaycomplex. In some embodiments the nucleic acid attached to the first highaffinity ligand is between 15 and 100 nucleotides in length, between 15and 80 nucleotides in length, between 15 and 50 nucleotides in length,between 5 and 40 nucleotides in length, between 15 and 30 nucleotides inlength, between 15 and 20 nucleotides in length, between 10 and 20nucleotides in length, or between 15 and 18 nucleotides in length. Insome embodiments the nucleic acid attached to the first high affinityligand is 15 nucleotides in length, 18, nucleotides in length, 20nucleotides in length, 30 nucleotides in length, 50 nucleotides inlength, 70 nucleotides in length, 80 nucleotides in length, or 87nucleotides in length.

Several embodiments of the present invention include a method of stablylinking an mRNA or modified mRNA, a NA linker operably linked to apuromycin or analogue or a small molecule, and a polypeptide encoded bythe mRNA together to form a linked mRNA-polypeptide complex.

In a preferred embodiment, the NA linker is used as a primer topolymerize a second strand of nucleic acid on the mRNA-polypeptidecomplex to form a nucleic acid duplex linked to the polypeptide. In apreferred embodiment, the polymerization is reverse transcription toform a DNA (or modified DNA) hybrid.

Several embodiments of the present invention include methods ofcomprising a plurality of distinct X-display complexes, providing aligand with a desired binding characteristic, contacting the complexeswith the ligand, removing unbound complexes, and recovering complexesbound to the ligand.

Several methods of the current invention involve the evolution ofnucleic acid molecules and/or proteins. In one embodiment, thisinvention comprises amplifying the nucleic acid component of therecovered complexes and introducing variation to the sequence of thenucleic acids. In other embodiments, the method further comprisestranslating polypeptides from the amplified and varied nucleic acids,linking them together using the nucleic acid/modified linkers, andcontacting them with the ligand to select another new population ofbound complexes. Several embodiments of the present invention useselected protein-mRNA complexes in a process of in vitro evolution,especially the iterative process in which the selected mRNA isreproduced with variation, translated and again connected to cognateprotein for selection.

Linker Moieties

In some embodiments, the present invention employs one or more linkermoieties (separate from the NA linker described above). In someembodiments linker moieties may be employed to connect a nucleic acid toa peptide acceptor. In other embodiments linker moieties may be used toconnect a high affinity ligand (e.g., biotin) or a ligand acceptor(e.g., streptavidin) to a peptide acceptor. In another embodiment,linker moieties may be used to connect a nucleic acid to a high affinityligand. As used herein, the term “linker moieties” may include one ormore linker moieties or subunits.

In some preferred embodiments the linker moieties are poly(alkyleneoxide) moieties, which are a genus of compounds having a polyetherbackbone. Poly(alkylene oxide) species of use in the present inventionmay include, for example, straight- and branched-chain species. Forexample, poly(ethylene glycol) is a poly(alkylene oxide) consisting ofrepeating ethylene oxide subunits, which may or may not includeadditional reactive, activatable or inert moieties at either terminus.Derivatives of straight-chain poly(alkylene oxide) species that areheterobifunctional are also known in the art. In some embodiments thelinker moiety may be composed of 5 to 50 subunits of poly(alkyleneoxide), 10 to 30 subunits of poly(alkylene oxide), 10 to 20 units ofpoly(alkylene oxide), 15 to 20 units of poly(alkylene oxide), or, insome embodiments, 18 subunits of poly(alkylene oxide). One of skill inthe art will appreciate that any number of linker moieties may be usedas long as it is still possible for the X-display complex to form.

A poly(ethylene glycol) linker is a moiety having a poly(ethyleneglycol) (“PEG”) backbone or methoxy-PEG (“mPEG”) backbone, including PEGand mPEG derivatives. A wide variety of PEG and mPEG derivatives areknown in the art and are commercially available. For example, Nektar,Inc. Huntsville, Ala., provides PEG and mPEG compounds useful as linkersor modifying groups optionally having nucleophilic reactive groups,carboxyl reactive groups, eletrophilically activated groups (e.g. activeesters, nitrophenyl carbonates, isocyanates, etc.), sulfhydryl selectivegroups (e.g. maleimide), and heterofunctional (having two reactivegroups at both ends of the PEG or mPEG), biotin groups, vinyl reactivegroups, silane groups, phospholipid groups, and the like.

In other embodiments the linker moieties may be nucleic acids or anyother linker recognized in the art. For example, Polysialic acids (PSAs)and PSA derivatives may be employed (see U.S. Pat. No. 5,846,951, U.S.Pat. Pub. No. US20080262209 and PCT App. WO2005/016973 andWO-A-01879221, which are all incorporated herein by reference in theirentirety.)

Although a preferred peptide acceptor is puromycin, other compounds thatact in a manner similar to puromycin may be used. Other possible choicesfor protein acceptors include pyrazolopyrimidine or any relatedderivatives and tRNA-like structures, and other compounds known in theart. Such compounds include, without limitation, any compound whichpossesses an amino acid linked to an adenine or an adenine-likecompound, such as the amino acid nucleotides, phenylalanyl-adenosine(A-Phe), tyrosyl adenosine (A-Tyr), and alanyl adenosine (A-Ala), aswell as amide-linked structures, such as phenylalanyl 3′ deoxy 3′ aminoadenosine, alanyl 3′ deoxy 3′ amino adenosine, and tyrosyl 3′ deoxy 3′amino adenosine; in any of these compounds, any of thenaturally-occurring L-amino acids or their analogs may be utilized. Inaddition, a combined tRNA-like 3′ structure-puromycin conjugate may alsobe used in the invention.

Translation Systems

Several embodiments of the invention utilize preferred methods whereinthe mRNA is translated in an in vitro translation system that lacksrelease factors. Thereby, the ribosome stalls at the stop codon,allowing time for the puromycin or analogue or a small molecule to bindcovalently or with high affinity to the polypeptide protein.

In some embodiments of the invention the mRNA is translated in an invitro translation system in which the function of at least one releasefactors is inhibited by release factor inhibitors. Suitable inhibitorsinclude, but are not limited to, anti-release factor antibodies,

As the method is preferably carried out using in vitro translationsystems, it is known in the art that modified amino acids can beincorporated into the translation machinery to create polypeptides withchemical modifications.

A variety of in vitro translation systems may be used such as areticulocyte lysate system, wheat germ extract system, or other suitablein vitro transcription system. In one preferred embodiment thePURESystem(cosmobio.co.jp/export_e/products/proteins/products_PGM_(—)20060907_(—)06.asp)is employed. The cell-free continuous-flow (CFCF) translation system ofSpirin et al. (1988) Science 242: 1162 may be used to increase totalyield of library members, or for convenience of use, if desired. Astatic in vitro protein synthesis system can be used. In this system,protein synthesis generally ceases after 1 h and thus limits the timeinterval for creation of the library. The advantage of CFCF technologyis that high level and long-term synthesis of protein should result in amuch larger and more diverse library of protein-RNA complexes. The CFCFtechnology has been described by Spirin and co-workers as a method forthe high-level synthesis of protein over an extended period of time, 24h or longer. In addition, CFCF technology results in fractionation ofthe newly-synthesized protein from the translational apparatus, and thusmakes it feasible to quickly sequester the protein-nucleic acidcomplexes. Other applications of CFCF technology include an efficientmethod for synthesizing peptides. For example, following theidentification of a peptide-fusion which binds to a target withhigh-affinity, the free peptide can be synthesized directly using CFCFtechnology and used in a binding assay.

Other cell-free techniques for linking a polynucleotide to a polypeptide(i.e., a phenotype) can also be used, e.g., Profusion™ (see, e.g., U.S.Pat. Nos. 6,348,315; 6,261,804; 6,258,558; and 6,214,553 which areincorporated herein by reference).

Suitable vectors can be chosen or constructed, containing appropriateregulatory sequences, including promoter sequences, terminatorsequences, polyadenylation sequences, enhancer sequences, marker genesand other sequences as appropriate. Vectors may be plasmids, viral e.g.‘phage, or phagemid, as appropriate for a particular expression or invitro translation system. For further details see, for example,Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al.,1989, Cold Spring Harbor Laboratory Press. Many known techniques andprotocols for manipulation of nucleic acid, for example in preparationof nucleic acid constructs, mutagenesis, sequencing, introduction of DNAinto cells and gene expression, and analysis of proteins, are describedin detail in Current Protocols in Molecular Biology, Second Edition,Ausubel et al. eds., John Wiley & Sons, 1992. The disclosures ofSambrook et al. and Ausubel et al. are incorporated herein by reference.

In some embodiments expression vectors such as plasmids or viral vectorsare not employed, e.g., when the DNA to be expressed exists only in aPCR amplified DNA strand.

In further embodiments, the methods the invention may employ methodsand/or compositions described in U.S. Pat. Nos. 7,078,197, 6,429,300,5,922,545, 7,195,880, 6,416,950, 6,602,685, 6,623,926, 6,951,725, or inU.S. patent application Ser. Nos. 11/543,316, 10/208,357, which are allincorporated herein by reference in their entirety.

In one preferred embodiment mRNA, containing an intact stop codon andregion of 3′ untranslated RNA sufficient for binding an oligonucleotideprimer, is translated in an in vitro translation system lacking releasefactors (e.g., PURESystem,cosmobio.co.jp/export_e/products/proteins/products_PGM_(—)20060907_(—)06.asp).Release factors trigger the hydrolysis of the ester bond inpeptidyl-tRNA and the release of the newly synthesized protein from theribosome. In the absence of the release factors, the ribosome will stallon the mRNA. Next a DNA oligonucleotide is added to the mix. Thisoligonucleotide is complementary to the 3′ end of the mRNA andfunctionalized with a linker which is capable of delivering a peptideacceptor species into the ribosome to form a covalent adduct with thebound translated protein. In some embodiments it is an NA linker whichis added. The site of attachment of the linker can be anywhere along theoligonucleotide with the exception of the 3′ end. The linker needs to beof sufficient length to reach into the ribosome. The species that formsthe adduct is preferably puromycin or pyrazolopyrimidine or any relatedderivatives.

Following the covalent addition of the linker to the nascent protein,EDTA is added to release the ribosomes, the mRNA-oligonucleotide-proteinspecies is subsequently isolated, and subjected to reverse transcriptionto create the DNA-protein fusion. Finally, the second strand of DNA isadded using any DNA polymerase. In such an embodiment, although themRNA-oligonucleotide (e.g., NA linker) species may be covalentlyattached, the NA linker is not required to be covalently attached to anyintermediary species in the X-display complex (e.g., ifbiotin/streptavidin is used to bridge the mRNA to the protein).

The resulting DNA-protein fusion can be used for in vivo or in vitroscreening or for diagnostic applications.

Uses

The methods and compositions of the present invention have commercialapplications in any area where protein technology is used to solvetherapeutic, diagnostic, or industrial problems. This X-displaytechnology is useful for improving or altering existing proteins as wellas for isolating new proteins with desired functions. These proteins maybe naturally-occurring sequences, may be altered forms ofnaturally-occurring sequences, or may be partly or fully syntheticsequences.

The methods of the invention can be used to develop or improvepolypeptides such as immunobinders, for example, antibodies, bindingfragments or analogs thereof, single chain antibodies, catalyticantibodies, VL and/or VH regions, Fab fragments, Fv fragments, Fab′fragments, Dabs, and the like. In some embodiments, the polypeptides tobe improved may be any polypeptide having an immunoglobulin orimmunoglobulin-like domain, for example Interferons, Protein A,Ankyrins, A-domains, T-cell receptors, Fibronectin III,gamma-Crystallin, antigen binding domains of MHC class molecules (e.g.,the alpha and beta antigen binding domains of CD8), Ubiquitin, membersof the immunoglobulin superfamily, and many others, as reviewed in Binz,A. et al. (2005) Nature Biotechnology 23:1257 and Barclay (2003) SeminImmunol. 15(4):215-223, which are incorporated herein by reference. Insome embodiments, like immunoglobulin libraries derived from the humanimmune repertoire, a single library uses many different V-regionsequences as scaffolds, but they all share the basic immunoglobulinfold. In some embodiments, the immunoglobulin or immunoglobulin-likefold is a barrel shaped protein structure comprising two β-sheetscomprising several (e.g., seven in the case of a light chain C-domain ofan IgG) anti-parallel β-strands held together by a disulfide bond.Accordingly, the improvement or selection of any immunoglobulin orimmunoglobulin-like protein, including portions or fragments thereof, iscontemplated.

In another application, the X-display technology described herein isuseful for the isolation of proteins with specific binding (for example,ligand binding) properties which may or may not have and immunoglobulinor immunoglobulin-like domain. Proteins exhibiting highly specificbinding interactions may be used as non-antibody recognition reagents,allowing X-Display technology to circumvent traditional monoclonalantibody technology. Antibody-type reagents isolated by this method maybe used in any area where traditional antibodies are utilized, includingdiagnostic and therapeutic applications.

In preferred embodiments, the methods will target the improvement ofimmunobinders, for example, regions of the variable region and/or CDRsof an antibody molecule, i.e., the structure responsible for antigenbinding activity which is made up of variable regions of two chains, onefrom the heavy chain (VH) and one from the light chain (VL). Once thedesired antigen-binding characteristics are identified, the variableregion(s) can be engineered into an appropriate antibody class such asIgG, IgM, IgA, IgD, or IgE. It is understood that the methods may beemployed to improve and/or select human immunobinders and/orimmunobinders from other species, e.g., any mammalian or non-mammalianimmunobinders, camelid antibodies, shark antibodies, etc.

The present invention may be used to improve human or humanizedantibodies (or fragments thereof) for the treatment of any of a numberof diseases. In this application, antibody libraries are developed andare screened in vitro, eliminating the need for techniques such ascell-fusion or phage display. In one important application, theinvention is useful for improving single chain antibody libraries (Wardet al., Nature 341:544 (1989); and Goulot et al., J. Mol. Biol. 213:617(1990)). For this application, the variable region may be constructedeither from a human source (to minimize possible adverse immunereactions of the recipient) or may contain a totally randomized cassette(to maximize the complexity of the library). To screen for improvedantibody molecules, a pool of candidate molecules are tested for bindingto a target molecule. Higher levels of stringency are then applied tothe binding step as the selection progresses from one round to the next.To increase stringency, conditions such as number of wash steps,concentration of excess competitor, buffer conditions, length of bindingreaction time, and choice of immobilization matrix may be altered.Single chain antibodies may be used either directly for therapy orindirectly for the design of standard antibodies. Such antibodies have anumber of potential applications, including the isolation ofanti-autoimmune antibodies, immune suppression, and in the developmentof vaccines for viral diseases such as AIDS.

As detailed below, a wide variety of antibody fragment and antibodymimetic technologies have now been developed and are widely known in theart. While a number of these technologies, such as domain antibodies,Nanobodies, and UniBodies make use of fragments of, or othermodifications to, traditional antibody structures, there are alsoalternative technologies, such as Adnectins, Affibodies, DARPins,Anticalins, Avimers, and Versabodies that employ binding structuresthat, while they mimic traditional antibody binding, are generated fromand function via distinct mechanisms. Some of these alternativestructures are reviewed in Gill and Damle (2006) 17: 653-658, whichincorporated herein by reference. All of the antibody derivatives andbinders mentioned above may be improved and/or selected by the methodsof the present invention. In some embodiments, methods known in the artto generate Nanobodies, UniBodies, Adnectins, Affibodies, DARPins,Anticalins, Avimers, and Versabodies may be used to discover an initialbinding protein which may then serve as the basis for the generation ofa library which may be produced and selected from according to themethods of the present invention. Alternatively, binders already knownin the art may be used directly to create new libraries for use with themethods described herein.

In some embodiments the methods described herein will target theimprovement of Domain Antibodies (dAbs). Domain Antibodies are thesmallest functional binding units of antibodies, corresponding to thevariable regions of either the heavy (VH) or light (VL) chains of humanantibodies. Domain Antibodies have a molecular weight of approximately13 kDa. Domantis has developed a series of large and highly functionallibraries of fully human VH and VL dAbs (more than ten billion differentsequences in each library), and uses these libraries to select dAbs thatare specific to therapeutic targets. In contrast to many conventionalantibodies, domain antibodies are well expressed in bacterial, yeast,and mammalian cell systems. Further details of domain antibodies andmethods of production thereof may be obtained by reference to U.S. Pat.Nos. 6,291,158; 6,582,915; 6,593,081; 6,172,197; 6,696,245; U.S. SerialNo. 2004/0110941; European patent application No. 1433846 and EuropeanPatents 0368684 & 0616640; WO05/035572, WO04/101790, WO04/081026,WO04/058821, WO04/003019 and WO03/002609, each of which is hereinincorporated by reference in its entirety.

In other embodiments the methods described herein will target theimprovement of nanobodies. Nanobodies are antibody-derived therapeuticproteins that contain the unique structural and functional properties ofnaturally-occurring heavy-chain antibodies. These heavy-chain antibodiescontain a single variable domain (VHH) and two constant domains (CH2 andCH3). Importantly, the cloned and isolated VHH domain is a perfectlystable polypeptide harbouring the full antigen-binding capacity of theoriginal heavy-chain antibody. Nanobodies have a high homology with theVH domains of human antibodies and can be further humanized without anyloss of activity. Importantly, Nanobodies have a low immunogenicpotential, which has been confirmed in primate studies with Nanobodylead compounds.

Nanobodies combine the advantages of conventional antibodies withimportant features of small molecule drugs. Like conventionalantibodies, Nanobodies show high target specificity, high affinity fortheir target and low inherent toxicity. However, like small moleculedrugs they can inhibit enzymes and readily access receptor clefts.Furthermore, Nanobodies are extremely stable, can be administered bymeans other than injection (see, e.g., WO 04/041867, which is hereinincorporated by reference in its entirety) and are easy to manufacture.Other advantages of Nanobodies include recognizing uncommon or hiddenepitopes as a result of their small size, binding into cavities oractive sites of protein targets with high affinity and selectivity dueto their unique 3-dimensional, drug format flexibility, tailoring ofhalf-life and ease and speed of drug discovery.

Nanobodies are encoded by single genes and are efficiently produced inalmost all prokaryotic and eukaryotic hosts, e.g., E. coli (see, e.g.,U.S. Pat. No. 6,765,087, which is herein incorporated by reference inits entirety), molds (for example Aspergillus or Trichoderma) and yeast(for example Saccharomyces, Kluyveromyces, Hansenula or Pichia) (see,e.g., U.S. Pat. No. 6,838,254, which is herein incorporated by referencein its entirety). The production process is scalable and multi-kilogramquantities of Nanobodies have been produced. Because Nanobodies exhibita superior stability compared with conventional antibodies, they can beformulated as a long shelf-life, ready-to-use solution. Accordingly, themethods of the present invention my be used to improve the affinity ofnanobodies for their target molecules.

Methods known in the art may be used to generate nanobodies (or otherbinders/immunobinders described herein). Such binders may then serve asthe basis for the generation of a library which may be produced andselected from according to the methods of the present invention. Forexample, the Nanoclone method (see, e.g., WO 06/079372, which is hereinincorporated by reference in its entirety) is a proprietary method forgenerating Nanobodies against a desired target, based on automatedhigh-throughout selection of B-cells and could be used in the context ofthe instant invention. The successful selection of nanobodies from theNanoclone method may provide an initial set of nanobodies which may befurther improved by the methods described herein.

In other embodiments the methods described herein will target theimprovement of UniBodies. Unibodies are another antibody fragmenttechnology, however this one is based upon the removal of the hingeregion of IgG4 antibodies. The deletion of the hinge region results in amolecule that is essentially half the size of traditional IgG4antibodies and has a univalent binding region rather than the bivalentbinding region of IgG4 antibodies. It is also well known that IgG4antibodies are inert and thus do not interact with the immune system,which may be advantageous for the treatment of diseases where an immuneresponse is not desired, and this advantage is passed onto UniBodies.For example, UniBodies may function to inhibit or silence, but not kill,the cells to which they are bound. Additionally, UniBody binding tocancer cells do not stimulate them to proliferate. Furthermore, becauseUniBodies are about half the size of traditional IgG4 antibodies, theymay show better distribution over larger solid tumors with potentiallyadvantageous efficacy. UniBodies are cleared from the body at a similarrate to whole IgG4 antibodies and are able to bind with a similaraffinity for their antigens as whole antibodies. Further details ofUniBodies may be obtained by reference to patent applicationWO2007/059782, which is herein incorporated by reference in itsentirety.

In other embodiments the methods described herein will target theimprovement of fibronectin or adnectin molecules. Adnectin molecules areengineered binding proteins derived from one or more domains of thefibronectin protein (see Ward M., and Marcey, D.,callutheran.edu/Academic_Programs/Departments/BioDev/omm/fibro/fibro.htm).Typically, fibronectin is made of three different protein modules, typeI, type II, and type III modules. For a review of the structure offunction of the fibronectin, see Pankov and Yamada (2002) J Cell Sci.;115(Pt 20):3861-3, Hohenester and Engel (2002) 21:115-128, and Lucena etal. (2007) Invest Clin. 48:249-262, which are incorporated herein byreference.

Depending on the originating tissue, fibronectin may contain multipletype III domains which may be denoted, e.g., ¹Fn3, ²Fn3, ³Fn3, etc. The¹⁰Fn3 domain contains an integrin binding motif and further containsthree loops which connect the beta strands. These loops may be thoughtof as corresponding to the antigen binding loops of the IgG heavy chain,and they may be altered by the methods discussed herein below to selectfibronectin and adnectin molecules that specifically bind a target ofinterest. Adnectin molecules to be improved may also be derived frompolymers of ¹⁰Fn3 related molecules rather than a simple monomeric ¹⁰Fn3structure.

Although the native ¹⁰Fn3 domain typically binds to integrin, ¹⁰Fn3proteins adapted to become adnectin molecules are altered so to bindantigens of interest. Accordingly, methods available to the skilledartisan may be used to create ¹⁰Fn3 variant and mutant sequences(thereby forming a library) which is compatible with the methods of thepresent invention. For example the alterations in the ¹⁰Fn3 may be madeby any method known in the art including, but not limited to, errorprone PCR, site-directed mutagenesis, DNA shuffling, or other types ofrecombinational mutagenesis which have been referenced herein. In oneexample, variants of the DNA encoding the ¹⁰Fn3 sequence may be directlysynthesized in vitro. Alternatively, a natural ¹⁰Fn3 sequence may beisolated or cloned from the genome using standard methods (as performed,e.g., in U.S. Pat. Application No. 20070082365, incorporated herein byreference), and then mutated using mutagenesis methods known in the art.

In one embodiment, a target protein, may be immobilized on a solidsupport, such as a column resin or a well in a microtiter plate. Thetarget is then contacted with a library of potential binding proteins orX-display complexes as described herein. The library may comprise ¹⁰Fn3clones or adnectin molecules derived from the wild type ¹⁰Fn3 bymutagenesis/randomization of the ¹⁰Fn3 sequence or bymutagenesis/randomization of the ¹⁰Fn3 loop regions (not the betastrands). The selection/mutagenesis process may be repeated untilbinders with sufficient affinity to the target are obtained. Adnectinmolecules for use in the present invention may be engineered using thePROfusion™ technology employed by Adnexus, a Briston-Myers Squibbcompany. The PROfusion™ technology was created based on the techniquesreferenced above (e.g., Roberts & Szostak (1997) 94:12297-12302).Methods of generating libraries of altered ¹⁰Fn3 domains and selectingappropriate binders which may be used with the present invention aredescribed fully in the following U.S. patent and patent applicationdocuments and are incorporated herein by reference: U.S. Pat. Nos.7,115,396; 6,818,418; 6,537,749; 6,660,473; 7,195,880; 6,416,950;6,214,553; 6,623,926; 6,312,927; 6,602,685; 6,518,018; 6,207,446;6,258,558; 6,436,665; 6,281,344; 7,270,950; 6,951,725; 6,846,655;7,078,197; 6,429,300; 7,125,669; 6,537,749; 6,660,473; and U.S. Pat.Application Nos. 20070082365; 20050255548; 20050038229; 20030143616;20020182597; 20020177158; 20040086980; 20040253612; 20030022236;20030013160; 20030027194; 20030013110; 20040259155; 20020182687;20060270604; 20060246059; 20030100004; 20030143616; and 20020182597.Also see the methods of the following references which are incorporatedherein by reference in their entirety: Lipov{hacek over (s)}ek et al.(2007) Journal of Molecular Biology 368: 1024-1041; Sergeeva et al.(2006) Adv Drug Deliv Rev. 58:1622-1654; Petty et al. (2007) TrendsBiotechnol. 25: 7-15; Rothe et al. (2006) Expert Opin Biol Ther.6:177-187; and Hoogenboom (2005) Nat Biotechnol. 23:1105-1116.

Additional molecules which can be improved using the methods of thepresent invention include, without limitation, human fibronectin modules¹Fn3-⁹Fn3 and ¹¹Fn3-¹⁷Fn3 as well as related Fn3 modules from non-humananimals and prokaryotes. In addition, Fn3 modules from other proteinswith sequence homology to ¹⁰Fn3, such as tenascins and undulins, mayalso be used. Other exemplary proteins having immunoglobulin-like folds(but with sequences that are unrelated to the V_(H) domain) includeN-cadherin, ICAM-2, titin, GCSF receptor, cytokine receptor, glycosidaseinhibitor, E-cadherin, and antibiotic chromoprotein. Further domainswith related structures may be derived from myelin membrane adhesionmolecule P0, CD8, CD4, CD2, class I MHC, T-cell antigen receptor, CD1,C2 and I-set domains of VCAM-1, I-set immunoglobulin fold ofmyosin-binding protein C, I-set immunoglobulin fold of myosin-bindingprotein H, I-set immunoglobulin-fold of telokin, telikin, NCAM,twitchin, neuroglian, growth hormone receptor, erythropoietin receptor,prolactin receptor, GC-SF receptor, interferon-gamma receptor,beta-galactosidase/glucuronidase, beta-glucuronidase, andtransglutaminase. Alternatively, any other protein that includes one ormore immunoglobulin-like folds may be utilized to create a adnectin-likebinding moiety which may be improved by the methods described herein.Such proteins may be identified, for example, using the program SCOP(Murzin et al., J. Mol. Biol. 247:536 (1995); Lo Conte et al., NucleicAcids Res. 25:257 (2000).

In other embodiments the methods of the present invention may beemployed to improve affibody molecules. Affibody molecules represent anew class of affinity proteins based on a 58-amino acid residue proteindomain, derived from one of the IgG-binding domains of staphylococcalprotein A. This three helix bundle domain has been used as a scaffoldfor the construction of combinatorial phagemid libraries, from whichAffibody variants that target the desired molecules can be selectedusing phage display technology (Nord K, Gunneriusson E, Ringdahl J,Stahl S, Uhlen M, Nygren P A, Binding proteins selected fromcombinatorial libraries of an α-helical bacterial receptor domain, NatBiotechnol 1997; 15:772-7. Ronmark J, Gronlund H, Uhlen M, Nygren P A,Human immunoglobulin A (IgA)-specific ligands from combinatorialengineering of protein A, Eur J Biochem 2002; 269:2647-55). Similarlibraries which may be produced by methods known in the art may beselected using the X-display technology described herein. The simple,robust structure of Affibody molecules in combination with their lowmolecular weight (6 kDa), make them suitable for a wide variety ofapplications, for instance, as detection reagents (Ronmark J, Hansson M,Nguyen T, et al, Construction and characterization of affibody-Fcchimeras produced in Escherichia coli, J Immunol Methods 2002;261:199-211) and to inhibit receptor interactions (Sandstorm K, Xu Z,Forsberg G, Nygren P A, Inhibition of the CD28-CD80 co-stimulationsignal by a CD28-binding Affibody ligand developed by combinatorialprotein engineering, Protein Eng 2003; 16:691-7). Further details ofAffibodies and methods of production thereof may be obtained byreference to U.S. Pat. No. 5,831,012 which is herein incorporated byreference in its entirety.

In other embodiments the methods of the present invention may beemployed to improve DARPins. DARPins (Designed Ankyrin Repeat Proteins)are one example of an antibody mimetic DRP (Designed Repeat Protein)technology that has been developed to exploit the binding abilities ofnon-antibody polypeptides. Repeat proteins such as ankyrin orleucine-rich repeat proteins, are ubiquitous binding molecules, whichoccur, unlike antibodies, ultra- and extracellularly. Their uniquemodular architecture features repeating structural units (repeats),which stack together to form elongated repeat domains displayingvariable and modular target-binding surfaces. Based on this modularity,combinatorial libraries of polypeptides with highly diversified bindingspecificities can be generated. This strategy includes the consensusdesign of self-compatible repeats displaying variable surface residuesand their random assembly into repeat domains.

DARPins can be produced in bacterial expression systems at very highyields and they belong to the most stable proteins known. Highlyspecific, high-affinity DARPins to a broad range of target proteins,including human receptors, cytokines, kinases, human proteases, virusesand membrane proteins, have been selected. DARPins having affinities inthe single-digit nanomolar to picomolar range can be obtained.

DARPins have been used in a wide range of applications, including ELISA,sandwich ELISA, flow cytometric analysis (FACS), immunohistochemistry(IHC), chip applications, affinity purification or Western blotting.DARPins also proved to be highly active in the intracellular compartmentfor example as intracellular marker proteins fused to green fluorescentprotein (GFP). DARPins were further used to inhibit viral entry withIC50 in the pM range. DARPins are not only ideal to blockprotein-protein interactions, but also to inhibit enzymes. Proteases,kinases and transporters have been successfully inhibited, most often anallosteric inhibition mode. Very fast and specific enrichments on thetumor and very favorable tumor to blood ratios make DARPins well suitedfor in vivo diagnostics or therapeutic approaches.

Additional information regarding DARPins and other DRP technologies canbe found in U.S. Patent Application Publication No. 2004/0132028 andInternational Patent Application Publication No. WO 02/20565, both ofwhich are hereby incorporated by reference in their entirety.

In other embodiments the methods of the present invention may beemployed to improve anticalins. Anticalins are an additional antibodymimetic technology, however in this case the binding specificity isderived from lipocalins, a family of low molecular weight proteins thatare naturally and abundantly expressed in human tissues and body fluids.Lipocalins have evolved to perform a range of functions in vivoassociated with the physiological transport and storage of chemicallysensitive or insoluble compounds. Lipocalins have a robust intrinsicstructure comprising a highly conserved β-barrel which supports fourloops at one terminus of the protein. These loops form the entrance to abinding pocket and conformational differences in this part of themolecule account for the variation in binding specificity betweenindividual lipocalins.

While the overall structure of hypervariable loops supported by aconserved β-sheet framework is reminiscent of immunoglobulins,lipocalins differ considerably from antibodies in terms of size, beingcomposed of a single polypeptide chain of 160-180 amino acids which ismarginally larger than a single immunoglobulin domain.

Lipocalins are cloned and their loops are subjected to engineering inorder to create Anticalins. Libraries of structurally diverse Anticalinshave been generated and Anticalin display allows the selection andscreening of binding function, followed by the expression and productionof soluble protein for further analysis in prokaryotic or eukaryoticsystems. Such Anticalin libraries may be employed in accordance with theX-display technology of the present invention. Studies have successfullydemonstrated that Anticalins can be developed that are specific forvirtually any human target protein can be isolated and bindingaffinities in the nanomolar or higher range can be obtained.

Anticalins can also be formatted as dual targeting proteins, so-calledDuocalin. A Duocalin binds two separate therapeutic targets in oneeasily produced monomeric protein using standard manufacturing processeswhile retaining target specificity and affinity regardless of thestructural orientation of its two binding domains. Anticalins selectedby the methods of the present invention may be assembled into Duocalinmolecules.

Modulation of multiple targets through a single molecule is particularlyadvantageous in diseases known to involve more than a single causativefactor. Moreover, bi- or multivalent binding formats such as Duocalinshave significant potential in targeting cell surface molecules indisease, mediating agonistic effects on signal transduction pathways orinducing enhanced internalization effects via binding and clustering ofcell surface receptors. Furthermore, the high intrinsic stability ofDuocalins is comparable to monomeric Anticalins, offering flexibleformulation and delivery potential for Duocalins. Additional informationregarding Anticalins can be found in U.S. Pat. No. 7,250,297 andInternational Patent Application Publication No. WO 99/16873, both ofwhich are hereby incorporated by reference in their entirety.

In other embodiments the methods of the present invention may beemployed to improve Avimers. Another antibody mimetic technology usefulin the context of the instant invention are Avimers. Avimers are evolvedfrom a large family of human extracellular receptor domains by in vitroexon shuffling and phage display, generating multidomain proteins withbinding and inhibitory properties. Linking multiple independent bindingdomains has been shown to create avidity and results in improvedaffinity and specificity compared with conventional single-epitopebinding proteins. Other potential advantages include simple andefficient production of multitarget-specific molecules in Escherichiacoli, improved thermostability and resistance to proteases. Avimers withsub-nanomolar affinities have been obtained against a variety oftargets, and these may be further improved by the methods describedherein.

Additional information regarding Avimers can be found in U.S. PatentApplication Publication Nos. 2006/0286603, 2006/0234299, 2006/0223114,2006/0177831, 2006/0008844, 2005/0221384, 2005/0164301, 2005/0089932,2005/0053973, 2005/0048512, 2004/0175756, all of which are herebyincorporated by reference in their entirety.

In other embodiments the methods of the present invention may beemployed to improve Versabodies. Versabodies are another antibodymimetic technology that could be used in the context of the instantinvention. Versabodies are small proteins of 3-5 kDa with >15%cysteines, which form a high disulfide density scaffold, replacing thehydrophobic core that typical proteins have. The replacement of a largenumber of hydrophobic amino acids, comprising the hydrophobic core, witha small number of disulfides results in a protein that is smaller, morehydrophilic (less aggregation and non-specific binding), more resistantto proteases and heat, and has a lower density of T-cell epitopes,because the residues that contribute most to MHC presentation arehydrophobic. All four of these properties are well-known to affectimmunogenicity, and together they are expected to cause a large decreasein immunogenicity.

The inspiration for Versabodies comes from the natural injectablebiopharmaceuticals produced by leeches, snakes, spiders, scorpions,snails, and anemones, which are known to exhibit unexpectedly lowimmunogenicity. Starting with selected natural protein families, bydesign and by screening the size, hydrophobicity, proteolytic antigenprocessing, and epitope density are minimized to levels far below theaverage for natural injectable proteins.

Given the structure of Versabodies, these antibody mimetics offer aversatile format that includes multi-valency, multi-specificity, adiversity of half-life mechanisms, tissue targeting modules and theabsence of the antibody Fc region. Furthermore, Versabodies may bemanufactured in E. coli at high yields, and because of theirhydrophilicity and small size, Versabodies are highly soluble and can beformulated to high concentrations. Versabodies are exceptionally heatstable (they can be boiled) and offer extended shelf-life. All of thequalities of the binders described herein (e.g., heat stability, saltstability, shelf life, immunogenicity, target affinity, etc.) may beimproved by the display and selection methods described herein(X-display methods).

Additional information regarding Versabodies can be found in U.S. PatentApplication Publication No. 2007/0191272 which is hereby incorporated byreference in its entirety.

In other embodiments the methods of the present invention may beemployed to improve SMIP™ molecules. SMIPs™ (Small ModularImmunoPharmaceuticals-Trubion Pharmaceuticals) engineered to maintainand optimize target binding, effector functions, in vivo half life, andexpression levels. SMIPS consist of three distinct modular domains.First they contain a binding domain which may consist of any proteinwhich confers specificity (e.g., cell surface receptors, single chainantibodies, soluble proteins, etc). Secondly, they contain a hingedomain which serves as a flexible linker between the binding domain andthe effector domain, and also helps control multimerization of the SMIPdrug. Finally, SMIPS contain an effector domain which may be derivedfrom a variety of molecules including Fc domains or other speciallydesigned proteins. The modularity of the design, which allows the simpleconstruction of SMIPs with a variety of different binding, hinge, andeffector domains, provides for rapid and customizable drug design. Thebinding domains of the SMIP™ molecules may also serve as the basis for alibrary suitable for display and selection according to the methodsdescribed herein (e.g., X-display methods).

More information on SMIPs, including examples of how to design them, maybe found in Zhao et al. (2007) Blood 110:2569-77 and the following U.S.Pat. App. Nos. 20050238646; 20050202534; 20050202028; 20050202023;20050202012; 20050186216; 20050180970; and 20050175614.

The detailed description of antibody fragment and antibody mimetictechnologies provided above is not intended to be a comprehensive listof all technologies that could be used in the context of the instantspecification. For example, and also not by way of limitation, a varietyof additional technologies including alternative polypeptide-basedtechnologies, such as fusions of complimentary determining regions asoutlined in Qui et al., Nature Biotechnology, 25(8) 921-929 (2007),which is hereby incorporated by reference in its entirety, could be usedin the context of the instant invention.

The X-Display complexes (e.g., nucleic acid-protein fusions orDNA-protein fusions) described herein may be used for any applicationpreviously described or envisioned for RNA-protein fusions. Commercialuses include the isolation of polypeptides with desired propertiesthrough in vitro evolution techniques (see, for example, Szostak et al.,U.S. Ser. No. 09/007,005 and U.S. Ser. No. 09/247,190; Szostak et al.,WO98/31700; Roberts & Szostak, Proc. Natl. Acad. Sci. USA (1997) vol.94, p. 12297-12302)), screening of cDNA libraries that are derived fromcellular mRNA (see, for example, Lipovsek et al., U.S. Ser. No.60/096,818, filed Aug. 17, 1998), and the cloning of new genes on thebasis of protein-protein interactions (Szostak et al., U.S. Ser. No.09/007,005 and U.S. Ser. No. 09/247,190; Szostak et al., WO98/31700), aswell as the use of these fusions in protein display experiments(Kuimelis et al. U.S. Ser. No. 60/080,686 and U.S. Ser. No. 09/282,734).The X-Display complexes (e.g., DNA-protein fusions) of the invention maybe used for any application previously described or envisioned forpreviously described display technologies such as those disclosed inU.S. Pat. Nos. 6,416,950; 6,429,300; 6,194,550; 6,207,446; and6,214,553, which are all incorporated herein by reference in theirentirety. These X-Display complexes (e.g., DNA-protein fusions) may beused for any appropriate therapeutic, diagnostic, or research purpose,particularly in the pharmaceutical and agricultural areas.

Isolation of New Catalysts

The present invention may also be used to select new catalytic proteins.In vitro selection and evolution has been used previously for theisolation of novel catalytic RNAs and DNAs, and, in the presentinvention, is used for the isolation of novel protein enzymes (anon-limiting example, merely provided for illustration, are enzymessuitable for carrying out the metabolism of input polymers into smallerand more immediately useful by-products, such as for convertingpolysaccharides into more useful biofuels). In one particular example ofthis approach, a catalyst may be isolated indirectly by selecting forbinding to a chemical analog of the catalyst's transition state. Inanother particular example, direct isolation may be carried out byselecting for covalent bond formation with a substrate (for example,using a substrate linked to an affinity tag) or by cleavage (forexample, by selecting for the ability to break a specific bond andthereby liberate catalytic members of a library from a solid support).

This approach to the isolation of new catalysts has at least twoimportant advantages over catalytic antibody technology (reviewed inSchultz et al., J. Chem. Engng. News 68:26 (1990)). First, in catalyticantibody technology, the initial pool is generally limited to theimmunoglobulin fold; in contrast, the starting library of X-displaycomplexes (DNA-protein fusions) may be either completely random or mayconsist, without limitation, of variants of known enzymatic structuresor protein scaffolds. In addition, the isolation of catalytic antibodiesgenerally relies on an initial selection for binding to transition statereaction analogs followed by laborious screening for active antibodies;again, in contrast, direct selection for catalysis is possible using anX-Display library approach, as previously demonstrated using RNAlibraries. In an alternative approach to isolating protein enzymes, thetransition-state-analog and direct selection approaches may be combined.

Enzymes obtained by this method are highly valuable. For example, therecurrently exists a pressing need for novel and effective industrialcatalysts that allow improved chemical processes to be developed. Amajor advantage of the invention is that selections may be carried outin arbitrary conditions and are not limited, for example, to in vivoconditions. The invention therefore facilitates the isolation of novelenzymes or improved variants of existing enzymes that can carry outhighly specific transformations (and thereby minimize the formation ofundesired byproducts) while functioning in predetermined environments,for example, environments of elevated temperature, pressure, or solventconcentration.

An In Vitro Interaction Trap

The X-display technology is also useful for screening cDNA libraries andcloning new genes on the basis of protein-protein interactions. By thismethod, a cDNA library is generated from a desired source (for example,by the method of Ausubel et al., Current Protocols in Molecular Biology,John Wiley & Sons and Greene Publishing Company, 1994; in particular seeChapter 5). Each of the candidate cDNAs may be formulated into anX-display complex (e.g., a DNA-protein fusion) using the techniquesdescribed herein, and the ability of these complexes (or improvedversions of the fusions) to interact with particular molecules is thentested.

The fact that the interaction step takes place in vitro allows carefulcontrol of the reaction stringency, using nonspecific competitor,temperature, and ionic conditions. Alteration of normal small moleculeswith non-hydrolyzable analogs (e.g., ATP vs. ATPgS) provides forselections that discriminate between different conformers of the samemolecule. This approach is useful for both the cloning and functionalidentification of many proteins since the nucleic acid sequence of theselected binding partner is associated and may therefore be readilyisolated. In addition, the technique is useful for identifying functionsand interactions of any human genes.

The method can also be used to develop or improve polypeptide ligandsfor improved half life, affinity, or solubility. The X-Display complexes(e.g., DNA-protein fusions) described herein may be used in anyselection method for desired proteins, including molecular evolution andrecognition approaches. Exemplary selection methods are described, forexample, in Szostak et al., U.S. Ser. No. 09/007,005 and U.S. Ser. No.09/247,190; Szostak et al., WO98/31700; Roberts & Szostak, Proc. Natl.Acad. Sci. USA (1997) vol. 94, p. 12297-12302; Lipovsek et al., U.S.Ser. No. 60/096,818 and U.S. Ser. No. 09/374,962; and Kuimelis et al.U.S. Ser. No. 60/080,686 and U.S. Ser. No. 09/282,734, all herebyincorporated by reference.

Library Generation, Screening and Affinity Maturation

In some embodiments peptide or gene libraries may be screened toidentify peptides having desired qualities (e.g., binding to aparticular antigen) or which have been improved or modified according tothe methods of the invention. In related embodiments, a particularpeptide produced or selected by the methods described herein may befurther altered by affinity maturation or mutagenesis, thereby producinga library of related peptides or nucleic acids. As such, one aspect ofthe invention may involve screening large libraries in order to identifypotential peptides (or nucleic acids encoding said peptides) which havedesirable qualities, such as the ability to bind an antigen, a higherbinding affinity, etc. Any methods for library generation and targetselection known in the art or described herein may be used in accordancewith the present invention.

Methods of library generation known in the art, in accordance with themethods described herein (e.g., for adding the appropriate tags,complementary sequences, etc.) may be employed to create librariessuitable for use with the methods described herein. Some methods forlibrary generation are described in U.S. Ser. Nos. 09/007,005 and09/247,190; Szostak et al., WO989/31700; Roberts & Szostak (1997)94:12297-12302; U.S. Ser. No. 60/110,549, U.S. Ser. No. 09/459,190, andWO 00/32823, which are incorporated herein by reference.

In one embodiment the library will comprise VH domains, preferably humanVH domains. Any cells may be used as a source for a library. In somepreferred embodiments the source of cells for the library may bePeripheral Blood Mononuclear Cells (PBMCs), splenocytes, or bone marrowcells (e.g., see FIG. 36 which describes the VH library diversity whichmay be obtained in a library from certain types of donor cells).

It will be understood by one of skill in the art that the methods of thepresent invention may be employed in an iterative fashion. For example,the nucleic acid or protein selected by one of the methods describedherein may serve as the basis for the generation of a new library fromwhich the process may begin again. An example of such a scheme isillustrated in FIG. 22, wherein the products of one round of selectionare used to regenerate a new library.

The X-display methodology described herein may be carried out underconditions such that intramolecular disulfide bonds are present in thepeptides during selections. In other embodiments, the formation ofdisulfide bonds may be prevented, if desired. A starting library may beobtained by, e.g., direct DNA synthesis, through in-vitro or in-vivomutagenesis, or any available starting library known in the art may beused. For example, the libraries and methods of making said librariesdescribed in the references cited herein, and in the followingreferences, which are all hereby incorporated in their entirety, may beused in accordance with the present invention: U.S. Pat. Nos. 5,922,545,7,074,557.

In preferred embodiments, the library is a VH or VL library. Table 2provides a non-limiting set of example primers which may be used toamplify VH domains.

TABLE 2 Exemplary primers for VH domain X-display library generation.R = A/G, Y = C/T, K = G/T, M = A/C, S = G/C, W = A/ Target regionPrimer details Primer Sequence SEQ ID No: C 1 IgM, cDNAACAGGAGACGAGGGGGAAAAG 1. synthesis, 21 mer C 1 IgG, cDNAGCCAGGGGGAAGACCGATGG 2. synthesis, 20 mer C 1 IgA, cDNAGAGGCTCAGCGGGAAGACCTT 3. synthesis, 22 mer G C 1 Vk, cDNACAACTGCTCATCAGATGGCGG 4. synthesis, 21 mer C 1 Vl, cDNACAGTGTGGCCTTGTTGGCTTG 5. synthesis, 21 mer C 2 PCR 3′ IgM, 21GGTTGGGGCGGATGCACTCCC 6. mer C 2 PCR 3′ IgG, 21 CGATGGGCCCTTGGTGGARGC 7.mer C 2 PCR 3′ IgA, 21 CTTGGGGCTGGTCGGGGATGC 8. mer C 2 PCR 3′Vk, 21 mer AGATGGTGCAGCCACAGTTCG 9. J 1-3C 2 PCR 3′ Vl, 42 merAGATGGTGCAGCCACAGTTCGTAGGACGGT 10. SASCTTGGTCCC J 7C 2 PCR 3′ Vl, 42 merAGATGGTGCAGCCACAGTTCGGAGGACGGT 11. CAGCTGGGTGCC VH1a PCR5′ VH1, 46CAATTACTATTTACAATTACAATGCAGGTK 12. mer CAGCTGGTGCAGTCTG VH1b PCR5′VH1, 46 CAATTACTATTTACAATTACAATGCAGGTCC 13. mer AGCTTGTGCAGTCTG VH1cPCR5′ VH1, 46 CAATTACTATTTACAATTACAATGCAGGTCC 14. mer AGCTGGTACAGTCTGVH1d PCR5′ VH1, 46 CAATTACTATTTACAATTACAATGCARATGC 15. merAGCTGGTGCAGTCTG VH2 PCR5′ VH2, 46 CAATTACTATTTACAATTACAATGCAGRTCA 16.mer CCTTGAAGGAGTCTG VH3a PCR5′ VH3, 46 CAATTACTATTTACAATTACAATGGARGTG17. mer CAGCTGGTGGAGTCTG VH3b PCR5′ VH3, 46CAATTACTATTTACAATTACAATGCAGGTG 18. mer CAGCTGGTGGAGTCTG VH3c PCR5′VH3, 46 CAATTACTATTTACAATTACAATGGAGGTG 19. mer CAGCTGTTGGAGTCTG VH4aPCR5′ VH4, 42 CAATTACTATTTACAATTACAATGCAGSTGC 20. mer AGCTGCAGGAG VH4bPCR5′ VH4, CAATTACTATTTACAATTACAATGCAGGTG 21. 45mer CAGCTACAGCAGTGG VH5PCR5′ VH5, CAATTACTATTTACAATTACAATGGARGTG 22. 46mer CAGCTGGTGCAGTCTG VH6PCR5′ VH6, CAATTACTATTTACAATTACAATGCAGGTA 23. 46mer CAGCTGCAGCAGTCAG VH7PCR5′ VH7, CAATTACTATTTACAATTACAATGCAGGTG 24. 46mer CAGCTGGTGCAATCTGlinkVk1a PCR5′ VK1, 48 GGCGGAGGTGGCTCTGGCGGTGGCGGATCG 25. merRACATCCAGATGACCCAG linkVk1b PCR5′ VK1, 48 GGCGGAGGTGGCTCTGGCGGTGGCGGATCG26. mer GMCATCCAGTTGACCCAG linkVk1c PCR5′ VK1, 48GGCGGAGGTGGCTCTGGCGGTGGCGGATCG 27. mer GCCATCCRGATGACCCAG linkVk1d PCR5′VK1, 48 GGCGGAGGTGGCTCTGGCGGTGGCGGATCG 28. mer GTCATCTGGATGACCCAGlinkVk2a PCR5′ VK2, 48 GGCGGAGGTGGCTCTGGCGGTGGCGGATCG 29. merGATATTGTGATGACCCAG linkVk2b PCR5′ VK2, 48 GGCGGAGGTGGCTCTGGCGGTGGCGGATCG30. mer GATRTTGTGATGACTCAG linkVk3a PCR5′ VK3, 48GGCGGAGGTGGCTCTGGCGGTGGCGGATCG 31. mer GAAATTGTGTTGACRCAG linkVk3a PCR5′VK3, 48 GGCGGAGGTGGCTCTGGCGGTGGCGGATCG 32. mer GAAATAGTGATGACGCAGlinkVk3a PCR5′ VK3, 48 GGCGGAGGTGGCTCTGGCGGTGGCGGATCG 33. merGAAATTGTAATGACACAG linkVk4 PCR5′ VK4, 48 GGCGGAGGTGGCTCTGGCGGTGGCGGATCG34. mer GACATCGTGATGACCCAG linkVk5 PCR5′ VK5, 48GGCGGAGGTGGCTCTGGCGGTGGCGGATCG 35. mer GAAACGACACTCACGCAG linkVk6a PCR5′VK6, 48 GGCGGAGGTGGCTCTGGCGGTGGCGGATCG 36. mer GAAATTGTGCTGACTCAGlinkVk6b PCR5′ VK6, 48 GGCGGAGGTGGCTCTGGCGGTGGCGGATCG 37. merGATGTTGTGATGACACAG linkVL1a PCR5′ VL1, 48 GGCGGAGGTGGCTCTGGCGGTGGCGGATCG38. mer CAGTCTGTGCTGACKCAG linkVL1b PCR5′ VL1, 48GGCGGAGGTGGCTCTGGCGGTGGCGGATCG 39. mer CAGTCTGTGYTGACGCAG linkVL2 PCR5′VL2, 48 GGCGGAGGTGGCTCTGGCGGTGGCGGATCG 40. mer CAGTCTGCCCTGACTCAGlinkVL3a PCR5′ VL3, 48 GGCGGAGGTGGCTCTGGCGGTGGCGGATCG 41. merTCCTATGWGCTGACTCAG linkVL3b PCR5′ VL3, 48 GGCGGAGGTGGCTCTGGCGGTGGCGGATCG42. mer TCCTATGAGCTGACACAG linkVL3c PCR5′ VL3, 48GGCGGAGGTGGCTCTGGCGGTGGCGGATCG 43. mer TCTTCTGAGCTGACTCAG linkVL3d PCR5′VL3, 48 GGCGGAGGTGGCTCTGGCGGTGGCGGATCG 44. mer TCCTATGAGCTGATGCAGlinkVL4 PCR5′ VL4, 48 GGCGGAGGTGGCTCTGGCGGTGGCGGATCG 45. merCAGCYTGTGCTGACTCAA linkVL5 PCR5′ VL5, 48 GGCGGAGGTGGCTCTGGCGGTGGCGGATCG46. mer CAGSCTGTGCTGACTCAG linkVL6 PCR5′ VL6, 48GGCGGAGGTGGCTCTGGCGGTGGCGGATCG 47. mer AATTTTATGCTGACTCAG linkVL7 PCR5′VL7, 48 GGCGGAGGTGGCTCTGGCGGTGGCGGATCG 48. mer CAGRCTGTGGTGACTCAGlinkVL8 PCR5′ VL8, 48 GGCGGAGGTGGCTCTGGCGGTGGCGGATCG 49. merCAGACTGTGGTGACCCAG linkVL4/9 PCR5′ VL4/9, 48GGCGGAGGTGGCTCTGGCGGTGGCGGATCG 50. mer CWGCCTGTGCTGACTCAG linkVL10 PCR5′VL10, 48 GGCGGAGGTGGCTCTGGCGGTGGCGGATCG 51. mer CAGGCAGGGCTGACTCAG

In preferred embodiments the nucleic acid constructs of the librarycontain the T7 promoter. The nucleic acids in the library may bemanipulated by any means known in the art to add appropriate promoters,enhancers, spacers, or tags which are useful for the production,selection, or purification of the nucleic acid, its translation product,or the X-display complex. For example, in some embodiments the sequencesin the library may include a TMV enhancer, sequences encoding a FLAGtag, an SA display sequence, or a polyadenylation sequence or signal. Insome embodiments the nucleic acid library sequences may further includea unique source tag to identify the source of the RNA or DNA sequence.In some embodiments the nucleic acid library sequences may include apool tag. A pool tag may be used to identify those sequence selectedduring a particular round of selection. This will allow, e.g., sequencesfrom multiple selection rounds to be pooled and sequenced in a singlerun without losing track of which selection round they originated from.

The double stranded DNA library is then transcribed in-vitro andassociated, as described herein, to a peptide acceptor. In one preferredembodiment, an NA linker or an NA linker attached to a high affinityligand (e.g., a biotinylated NA linker) is then annealed (e.g., DDB-1).In some embodiments, the NA linker is photocrosslinked to the mRNA. Inparticular embodiments a ligand acceptor, e.g., streptavidin, is thenloaded. In further embodiments a second high affinity ligand which isattached to a peptide acceptor is bound to the streptavidin. In someembodiments the second high affinity ligand/peptide acceptor is abiotin-puromycin linker, e.g., BPP.

in vitro translation may then be carried out wherein the peptideacceptor reacts with the nascent translation product.

The result, after purification, is a library of peptide-nucleic acidX-display complexes. Such X-display complexes may then undergo reversetranscription after, in preferred embodiments, being purified. TheX-display complexes may be purified by any method known in the art,e.g., by affinity chromatography, column chromatography, densitygradient centrifugation, affinity tag capture, etc. In a preferredembodiment an oligo-dT cellulose purification is employed wherein theX-display complex has been designed to include an mRNA with a poly-Atail. In such embodiments oligo-dT is covalently bound to the cellulosein the column or purification device. The oligo-dT participates incomplementary base pairing with the poly-A tail of the mRNA in theX-display complex, thereby impeding its progress in through thepurification device. The X-display complex may later be eluted withwater or buffer.

Reverse transcription generates a cDNA/RNA hybrid, which, in preferredembodiments is noncovalently linked to the transcribed peptide throughassociation with the NA linker, the high affinity ligand, the ligandacceptor, the peptide acceptor (possibly linked to a second highaffinity ligand), or some operable combination thereof.

The resulting, purified X-display complex may then be treated with RNAseto degrade the remaining mRNA, followed by second strand DNA synthesisto generate a complete cDNA. Note that, in a preferred embodiment, thenucleic acids in the NA linker may serve as a primer for reversetranscription. Accordingly, the cDNA remains attached to the highaffinity ligand and part of the X-display complex.

The X-display complex may be further purified if, as is contemplated,the X-display complex is engineered to contain a tag. Any tag known inthe art may be used to purify the X-display complex. For example, it ispossible to use a FLAG tag, myc tac, Histidine tag (His tag), or HA tag,among others. In preferred embodiments a sequence encoding a FLAG tag isengineered into the original DNA sequence such that the finaltranscribed protein contains the FLAG tag.

The resulting X-display complex is then selected for by using anyselection method known in the art. In preferred embodiments affinityselection is used. For example, the desired binding target or antigenmay be immobilized on a solid support for use in an affinity column.Examples of methods useful in affinity chromatography are described inU.S. Pat. Nos. 4,431,546, 4,431,544, 4,385,991, 4,213,860, 4,175,182,3,983,001, 5,043,062, which are all incorporated herein by reference intheir entirety. Binding activity can be evaluated by standardimmunoassay and/or affinity chromatography. Screening of X-displaycomplexes for catalytic function, e.g., proteolytic function can beaccomplished using a standard hemoglobin plaque assay as described, forexample, in U.S. Pat. No. 5,798,208. Determining the ability ofcandidate peptides (e.g., antibodies, single chain antibodies, etc.) tobind therapeutic targets can be assayed in vitro using, e.g., a Biacoreinstrument, which measures binding rates of an antibody to a giventarget or antigen.

In preferred embodiments, the selected X-display complexes may beidentified by sequencing of the DNA component. Any sequencing technologyknown in the art may be employed, e.g., 454 Sequencing, Sangersequencing, sequencing by synthesis, or the methods described in U.S.Pat. Nos. 5,547,835, 5,171,534, 5,622,824, 5,674,743, 4,811,218,5,846,727, 5,075,216, 5,405,746, 5,858,671, 5,374,527, 5,409,811,5,707,804, 5,821,058, 6,087,095, 5,876,934, 6,258,533, 5,149,625 whichare all incorporated herein by reference in their entirety.

In some embodiments the selection may be performed multiple times toidentify higher affinity binders, and may further be implemented withcompetitive binders or more stringent washing conditions. One of skillin the art will appreciate that variants of the procedure describedherein may be employed.

In a preferred embodiment, the methods of the present invention arecarried out as depicted in FIG. 14.

In other preferred embodiments the X-display complex is designed asdepicted in FIG. 13 or FIG. 16.

Pharmaceutical Compositions Containing the Peptides or Mimetics Thereofof the Invention

In another aspect, the present invention provides a composition, e.g., apharmaceutical composition, containing one or a combination of thepeptides (e.g., two or more different peptides) generated by the methodsof the invention, formulated together with a pharmaceutically acceptablecarrier. Pharmaceutical compositions of the invention also can beadministered in combination therapy, i.e., combined with other agents.For example, the combination therapy can include a peptide or mimeticthereof of the invention combined with other appropriate pharmaceuticalagents useful for treating a particular disease or indication.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion). Depending onthe route of administration, the active compound, i.e., the peptide ormimetic thereof of the invention may be coated in a material to protectthe compound from the action of acids and other natural conditions thatmay inactivate the compound.

The pharmaceutical compounds of the invention may include one or morepharmaceutically acceptable salts. A “pharmaceutically acceptable salt”refers to a salt that retains the desired biological activity of theparent compound and does not impart any undesired toxicological effects(see e.g., Berge, S. M., et al. (1977) J. Pharm. Sci. 66:1-19). Examplesof such salts include acid addition salts and base addition salts. Acidaddition salts include those derived from nontoxic inorganic acids, suchas hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic,phosphorous and the like, as well as from nontoxic organic acids such asaliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoicacids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromaticsulfonic acids and the like. Base addition salts include those derivedfrom alkaline earth metals, such as sodium, potassium, magnesium,calcium and the like, as well as from nontoxic organic amines, such asN,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,choline, diethanolamine, ethylenediamine, procaine and the like.

In a particular embodiment the a peptides or mimetic thereof selected bymethods of the invention may be dissolved in water with sodium chlorideto achieve physiological isotonic salt conditions.

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures, and by the inclusion of various antibacterial and antifungalagents, for example, paraben, chlorobutanol, phenol sorbic acid, and thelike. It may also be desirable to include isotonic agents, such assugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositions ofthe invention is contemplated. Supplementary active compounds can alsobe incorporated into the compositions.

The solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings and other coatings well-known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes. The peptides can also be inmicro-encapsulated form, if appropriate, with one or more excipients.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

A composition formulated as a solution may be made suitable foradministration by dropper into the eye, e.g., by preparing the solutionto contain the appropriate amount of salts.

Liposomes containing a peptide or mimetic thereof selected by methods ofthe present invention can be prepared in accordance with any of the wellknown methods such as described by Epstein et al. (Proc. Natl. Acad.Sci. USA 82: 3688-3692 (1985)), Hwang et al. (Proc. Natl. Acad. Sci. USA77: 4030-4034 (1980)), EP 52,322, EP 36,676; EP 88,046; EP 143,949; EP142,641; Japanese Pat. Appl. 83-118008, and EP 102,324, as well as U.S.Pat. Nos. 4,485,045 and 4,544,545, the contents of which are herebyincorporated by reference in their entirety. Liposomes may be small(about 200-800 Angstroms) unilamellar type in which the lipid content isgreater than about 10 mol. percent cholesterol, preferably in a range of10 to 40 mol. percent cholesterol, the selected proportion beingadjusted for optimal peptide therapy. However, as will be understood bythose of skill in the art upon reading this disclosure, phospholipidvesicles other than liposomes can also be used.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

The amount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thesubject being treated, and the particular mode of administration. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will generally be that amountof the composition which produces a therapeutic effect. Generally, outof one hundred percent, this amount will range from about 0.01 percentto about ninety-nine percent of active ingredient, preferably from about0.1 percent to about 70 percent, most preferably from about 1 percent toabout 30 percent of active ingredient in combination with apharmaceutically acceptable carrier.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

For administration of the peptide or mimetic thereof of the invention,the dosage ranges from about 0.0001 to 100 mg/kg, and more usually 0.01to 5 mg/kg, of the host body weight. For example dosages can be 0.3mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kgbody weight or 10 mg/kg body weight or within the range of 1-10 mg/kg.An exemplary treatment regime entails administration once per week, onceevery two weeks, once every three weeks, once every four weeks, once amonth, once every 3 months or once every three to 6 months. Preferreddosage regimens for a moiety of the invention include 1 mg/kg bodyweight or 3 mg/kg body weight via intravenous administration, with theantibody being given using one of the following dosing schedules: (i)every four weeks for six dosages, then every three months; (ii) everythree weeks; (iii) 3 mg/kg body weight once followed by 1 mg/kg bodyweight every three weeks.

Alternatively, the peptide or mimetic thereof selected by the methods ofthe invention can be administered as a sustained release formulation, inwhich case less frequent administration is required. Dosage andfrequency vary depending on the half-life of the administered substancein the patient. The dosage and frequency of administration can varydepending on whether the treatment is prophylactic or therapeutic. Inprophylactic applications, a relatively low dosage is administered atrelatively infrequent intervals over a long period of time. Somepatients continue to receive treatment for the rest of their lives. Intherapeutic applications, a relatively high dosage at relatively shortintervals is sometimes required until progression of the disease isreduced or terminated, and preferably until the patient shows partial orcomplete amelioration of symptoms of disease. Thereafter, the patientcan be administered a prophylactic regime.

Actual dosage levels of the active ingredients and small molecules inthe pharmaceutical compositions of the present invention may be variedso as to obtain an amount of the active ingredient which is effective toachieve the desired therapeutic response for a particular patient,composition, and mode of administration, without being toxic to thepatient. The selected dosage level will depend upon a variety ofpharmacokinetic factors including the activity of the particularcompositions of the present invention employed, or the ester, salt oramide thereof, the route of administration, the time of administration,the rate of excretion of the particular compound being employed, theduration of the treatment, other drugs, compounds and/or materials usedin combination with the particular compositions employed, the age, sex,weight, condition, general health and prior medical history of thepatient being treated, and like factors well known in the medical arts.

A “therapeutically effective dosage” of a peptide or mimetic thereofselected by the methods of the invention preferably results in adecrease in severity of disease symptoms, an increase in frequency andduration of disease symptom-free periods, or a prevention of impairmentor disability due to the disease affliction. For example, for thetreatment of tumors, a “therapeutically effective dosage” preferablyinhibits cell growth or tumor growth by at least about 10% or 20%, morepreferably by at least about 40%, even more preferably by at least about60%, and still more preferably by at least about 80% relative tountreated subjects. The ability of a compound to inhibit tumor growthcan be evaluated in an animal model system predictive of efficacy inhuman tumors. Alternatively, this property of a composition can beevaluated by examining the ability of the compound to inhibit, suchinhibition in vitro by assays known to the skilled practitioner. Atherapeutically effective amount of a therapeutic compound can decreasetumor size, or otherwise ameliorate symptoms in a subject. One ofordinary skill in the art would be able to determine such amounts basedon such factors as the subject's size, the severity of the subject'ssymptoms, and the particular composition or route of administrationselected.

A composition of the present invention can be administered via one ormore routes of administration using one or more of a variety of methodsknown in the art. As will be appreciated by the skilled artisan, theroute and/or mode of administration will vary depending upon the desiredresults. Preferred routes of administration for binding moieties of theinvention include intravenous, intramuscular, intradermal,intraperitoneal, subcutaneous, spinal or other parenteral routes ofadministration, for example by injection or infusion. The phrase“parenteral administration” as used herein means modes of administrationother than enteral and topical administration, usually by injection, andincludes, without limitation, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural andintrasternal injection and infusion.

Alternatively, a peptide or mimetic thereof of the invention can beadministered via a non-parenteral route, such as a topical, epidermal ormucosal route of administration, for example, intranasally, orally,vaginally, rectally, sublingually or topically.

The active compounds can be prepared with carriers that will protect thecompound against rapid release, such as a controlled releaseformulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

Therapeutic compositions can be administered with medical devices knownin the art. For example, in a preferred embodiment, a therapeuticcomposition of the invention can be administered with a needlelesshypodermic injection device, such as the devices disclosed in U.S. Pat.Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824;or 4,596,556. Examples of well-known implants and modules useful in thepresent invention include: U.S. Pat. No. 4,487,603, which discloses animplantable micro-infusion pump for dispensing medication at acontrolled rate; U.S. Pat. No. 4,486,194, which discloses a therapeuticdevice for administering medicants through the skin; U.S. Pat. No.4,447,233, which discloses a medication infusion pump for deliveringmedication at a precise infusion rate; U.S. Pat. No. 4,447,224, whichdiscloses a variable flow implantable infusion apparatus for continuousdrug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drugdelivery system having multi-chamber compartments; and U.S. Pat. No.4,475,196, which discloses an osmotic drug delivery system. Thesepatents are incorporated herein by reference. Many other such implants,delivery systems, and modules are known to those skilled in the art.

The present invention is further illustrated in the following examples,which should not be construed as limiting.

EXEMPLIFICATION

Throughout the examples, the following materials and methods were usedunless otherwise stated.

Materials and Methods

In general, the practice of the present invention employs, unlessotherwise; indicated, conventional techniques of chemistry, molecularbiology, recombinant DNA technology, PCR technology, immunology(especially, e.g. antibody technology), expression systems (e.g.,cell-free expression, phage display, ribosome display, and Profusion),and any necessary cell culture that are within the skill of the art andare explained in the literature. See, e.g. Sambrook, Fritsch andManiatis, Molecular Cloning: Cold Spring Harbor Laboratory Press (1989);DNA Cloning, Vols. 1 and 2, (D. N. Glover, Ed. 1985); OligonucleotideSynthesis (M. J. Gait, Ed. 1984); PCR Handbook Current Protocols inNucleic Acid Chemistry, Beaucage, Ed. John Wiley & Sons (1999) (Editor);Oxford Handbook of Nucleic Acid Structure, Neidle, Ed., Oxford UnivPress (1999); PCR Protocols: A Guide to Methods and Applications, Inniset al., Academic Press (1990); PCR Essential Techniques: EssentialTechniques, Burke, Ed., John Wiley & Son Ltd (1996); Ike PCR Technique:RT-PCR, Siebert, Ed., Eaton Pub. Co. (1998); Antibody EngineeringProtocols (Methods in Molecular Bioogy), 510, Paul, S., Humana Pr(1996); Antibody Engineering: A Practical Approach (Practical ApproachSeries, 169), McCafferty, Ed., Irl Pr (1996); Antibodies: A LaboratoryManual, Harlow et al., C.S.H.L. Press, Pub. (1999); Current Protocols inMolecular Biology, eds. Ausubel et al., John Wiley & Sons (1992);Large-Scale Mammalian Cell; Culture Technology, Lubiniecki, A., Ed.,Marcel Dekker, Pub., (1990).

The compositions and methods of the invention can, in some embodiments,be used in conjunction with those of any other art recognized in vitrodisplay system, for example, those described in U.S. Pat. Nos.7,195,880; 6,951,725; 7,078,197; 7,022,479, 6,518,018; 7,125,669;6,846,655; 6,281,344; 6,207,446; 6,214,553; 6,258,558; 6,261,804;6,429,300; 6,489,116; 6,436,665; 6,537,749; 6,602,685; 6,623,926;6,416,950; 6,660,473; 6,312,927; 5,922,545; 6,194,550; 6,207,446;6,214,553; and 6,348,315., which are hereby incorporated by reference.

EXAMPLES

The present disclosure is further illustrated by the following examples,which should not be construed as further limiting.

Example 1 Design and Construction of Naïve Antibody Libraries Sources ofCells

mRNA was obtained from whole bone marrow (10 donors), spleenocytes (13donors) and peripheral mononuclear cells (601 donors) of total 624different healthy individuals, to ensure the diversity of the library.The calculated diversity of VH library is 10⁹-10¹⁹ and VL library is10⁶-10⁷.

Library Construction RT-PCR was Used for VH and VL Library Construction.

First strand cDNA was synthesized using specific primers from the Hchain constant regions of IgM, IgG and IgA (C 1, C 1 and C 1), and Lchain constant regions of kappa and lambda (C 1 and C).

For variable H chain library construction, multiple sense primers(degenerate primers) were designed from the FR1 regions of VH1-7 familymembers with an upstream UTR sequence (VH1-7UTR). The anti-sense primersfor VH were designed from the constant regions nested to the primers forcDNA synthesis (C 2, C 2 and C 2). For variable light chain libraryconstruction, multiple sense primers were designed from the V and V FR1regions of each family with an upstream 12 amino acids of the linkersequence to allow for single chain Fv shuffling. The anti-sense primersfor and gene amplification were designed from the constant regionsnested to C 1 (C 2) or J with the same C 2 downstream (J C 2). Theassembled scFv of all IgM and IgG families will have the same C 2sequences at the downstream end if needed.

For VH library construction, PCR was performed with C 2, C 2, C 2 andVH1-7 UTR as individual pairs for all three sources of B cells and gelpurified. After purification, individual families amplified from 3sources were pooled and generated 7 (VH1-7) IgM libraries, 7 (VH1-7) IgGlibraries and 7 IgA libraries (VH1-7). For VL library construction, PCRwas performed with C 2 and V or J C 2 mix and V for 3 sources of Bcells. After gel purification, V and V libraries from different sourceswere pooled to generate V and V libraries.

Library Modification

IgM, IgG, IgA VH libraries and VL libraries were also modified to carrythe in vitro transcription, translation signal sequences at 5′ end andtag sequences at 3′ end. These VH libraries are ready to be made intostreptavidin display libraries.

Oligo Sequences for Library Construction

The oligonucleotides primers set forth in Table 2 were used to generateVH and VL X-display libraries.

Example 2 Production and Use of Streptavidin Display Libraries

This example describes the production and use of the streptavidindisplay libraries of the invention

Materials and Methods

In general, the practice of the present invention may employ, unlessotherwise indicated, conventional techniques of chemistry, molecularbiology, recombinant DNA technology, immunology (especially, e.g.,antibody technology), and standard techniques of polypeptidepreparation. See, e.g., Sambrook, Fritsch and Maniatis, MolecularCloning: Cold Spring Harbor Laboratory Press (1989); AntibodyEngineering Protocols (Methods in Molecular Biology), 510, Paul, S.,Humana Pr (1996); Antibody Engineering: A Practical Approach (PracticalApproach Series, 169), McCafferty, Ed., Irl Pr (1996); Antibodies: ALaboratory Manual, Harlow et al., C.S.H.L. Press, Pub. (1999); andCurrent Protocols in Molecular Biology, eds. Ausubel et al., John Wiley& Sons (1992).

Buffers

10× chemical ligation buffer: 250 mM Tris pH7 and 1M NaCl1× oligo dT binding buffer: 100 mM Tris pH8, 1M NaCl, and 0.05% TritonX-1002× oligo dT binding buffer: 200 mM Tris pH8, 2M NaCl, and 0.1% TritonX-1001× flag binding buffer: 50 mM HEPES, 150 mM NaCl, and 0.025% TritonX-100 or

1×PBS and 0.025% Triton X-100

5× flag binding buffer: 250 mM HEPES, 750 mM NaCl, and 0.125% TritonX-100 or 5×PBS and 0.125% Triton X-100

Source of Used Reagents Megascript T7: Ambion (Austin, Tex.) PH 1334

Retic lysate IVT: Ambion (Austin, Tex.) PH1200UC Master mix-met: Ambion (Austin, Tex.) PH 1223G

Superscript II RNaseH-RT: Invitrogen (Carlsbad, Calif.) #18064-014

NAP-25 column: Amersham Pharmacia Biotech (Sunnyvale, Calif.)#17-0852-01Oligo dT cellulose Type 7: Amersham Pharmacia Biotech (Sunnyvale,Calif.) #27-5543-03Anti-flag M2 agarose affinity gel: Sigma (St. Louis, Mo.) #A2220Flag peptide: Sigma (St. Louis, Mo.) #F3290dNTP: Amersham Pharmacia Biotech (Sunnyvale, Calif.) #27-2035-01Herculase Hotstart DNA polymerase: Stratagene (La Jolla, Calif.)600312-51Mini spin column: Biorad (Hercules, Calif.) #732-6204

Ultrapure BSA: Ambion (Foster City, Calif.) #2616

Sheared salmon sperm DNA: Ambion (Foster City, Calif.) #9680

Linkers: PBI, DDB, BPP: Trilink BioTechnologies, Inc. (San Diego,Calif.) H₂O: OmniPur (Gibbstown, N.J.) #9610 2M KCl: Usb (Cleveland,Ohio) #75896 1M MgCl2: Usb (Cleveland, Ohio) #78641 0.5M EDTA: Usb(Cleveland, Ohio) #15694

1M Tris pH8: Usb (Cleveland, Ohio) #226381M Tris pH7: Usb (Cleveland, Ohio) #22637

5M NaCl: Usb (Cleveland, Ohio) #75888 1M HEPES: Usb (Cleveland, Ohio)#16924

Qiaquick gel extraction kit: Qiagen (Valencia, Calif.) #287066% TBE-Urea gels: Invitrogen (Carlsbad, Calif.) EC6865BOX

4-16% NativePAGE Gel: Invitrogen (Carlsbad, Calif.) Special ReagentsPreparation

Oligo dT Cellulose (Final Concentration: 100 mg/ml)

2.5 g of oligo dT cellulose were mixed with 25 ml of 0.1N NaOH in a 50ml tube. After spinning of the mixture at 1500 rpm for 3 minutes, theresulting supernatant was discarded. The resulting pellet containing theoligo dT cellulose was washed with 25 ml of 1× oligo dT binding bufferand then precipitated again by spinning at 1500 rpm for 3 minutes. Theresulting supernatant was again separated from the pellet and discarded.This wash step was repeated for 3 more times. After the last time ofwashing, the pH of the resulting supernatant was measured. The pH shouldbe the same as wash buffer (˜pH 8.5). Then the pellet containing theoligo dT cellulose was separated from the supernatant and re-suspendedin 25 ml of 1× oligo dT binding buffer before storing at 4 C.

M2 Agarose Preparation

25 ml of M2 agarose slurry was transferred into a 50 ml of tube. Afterspinning for 5 minutes at 1000 RPM in a Beckman centrifuge, thesupernatant was separated and discarded. The resulting pellet containingM2 agarose was re-suspended with one column volume of Glycine 10 mM pH3.5 and spilled for 5 minutes at 1000 RPM. The resulting supernatant wasagain discarded. The agarose pellet was then re-suspended with onecolumn volume of 1× flag binding buffer. The mixture was spilled for 5minutes at 1000 RPM and the resulting supernatant was discarded. Thiswash step was repeated for 3 times and then the pellet was re-suspendedwith one column volume of 1× binding buffer (with 1 mg/ml of BSA and 100μg/ml of sssDNA). The mixture was tumbled for 1 hour or overnight at 4°C. before being separated in aliquots in 2 ml fractions and stored at 4°C.

Amplification of the VH Library

An exemplary method for amplifying the VH library is given below:

Primers: 5′ Primer: S7 (T7TMVUTR) (SEQ ID No: 52)5′-TAATACGACTCACTATAGGGACAATTACTATTTACAATTACA-3′ 5′Primer: S7a (T7TMVUTR AUG) (SEQ ID No: 53)5′-TAATACGACTCACTATAGGGACAATTACTATTTACAATTACAATG-3′ 3′Primer: XB-S5-1 (Cμ2flagA20 + SA display added sequence) (SEQ ID No: 54)5′-TTTTTTTTTTTTTTTTTTTTAAAT AGC GGA TGC TAA GGACGA CTTGTCGTCGTCGTCCTTGTAGTC GGTTGGGGCGGATGCACTCC C-3′Reverse Frame 1 (translation of the coding strandcomplementary to the primer): (SEQ ID Nos: 55 and 92)1 G S A S A P T D Y K D D D D K S S L A S A I (STOP) K K K K K K

Reaction set up:

10X Herculase Buffer (Strategene) 50 μl 10 mM dNTP 10 μl S7 (5 μM) 20 μlS5-1 (5 μM) 20 μl VH library (1-6 families) 40 ng H2O X μl Herculase(Strategene) 10 μl 500 μl (50 μl/reaction)

Temperature cycling program:

95 C./3 minutes  1 cycle 95 C./30 seconds 50 C./30 seconds {closeoversize bracket} 20 cycles 72 C./1 minutes 72 C./10 minutes  1 cycleTm of 50 C is approximate and depends on a choice of PCR primers.

Library Purification:

Amplified VH library DNAs were separated on 2% agarose gel. The 400 bpband was cut out under UV light for DNA extraction from the gel sliceusing Qiaquick Gel Extraction Kit (Qiagen #28706, Valencia, Calif.) andreconstitution in 300 μl H₂O. 5 μL of purified DNA was applied forelectrophoresis on a 2% E-gel. The recovery rate was expected to be˜80%.

Transcription

Reaction Set-Up (MEGAscript Kit™, Ambion PH 1334, Foster City, Calif.)

For first round of library production, a full scale of RNA transcription(500 μl) is recommended. The reaction volume can be scaled down at laterrounds.

PCR product (5-10 μg) 200 μL  10X reaction buffer 50 μL ATP (75 mM) 50μL CTP (75 mM) 50 μL GTP (75 mM) 50 μL UTP (75 mM) 50 μL T7 polymerase50 μL Total: 500 μL 

Incubation

The reaction mixture as above was incubated at 37° C. for 1-2 hrs or,preferably, up to overnight.

Purification

An exemplary purification method is fractionation on a NAP-25 column,which is given below. NAP 10 and NAP 5 columns can be used forpurification of small scale production, in which cases the fractionvolume should be changed accordingly.

For a purification process using a NAP-25 column, 25 μL Dnase I can beadded to every 500 μl transcription reaction. The mixture will beincubated at 37° C. for 15 minutes before phenol extraction, when 500 μLphenol-chloroform-isoamylalcohol is added to the mixture. The mixturecan be then vortexed for 30 seconds. After microfuging the mixture for 5minutes, the aqueous phase can be then recovered. Before loading of thisaqueous phase, a NAP-25 column can be pre-washed by letting 10 mL dH₂Odrip through the column. Then the extracted transcription mix can beloaded with to washed column and run into the column. 800 μL dH₂O canthen be added to column and the run-through can be collected. Thiselution process can be repeated for five times (E1-E5) and the RNAconcentration in collected sample can be measured by A₂₆₀ on aspectrophotometer. 2 μL of sample can be applied for electrophoresis ona 2% E-gel to QC. The fraction containing most RNA can be used forligation (E3 contains little amount, E4 is the peak fraction, E5contains the mixture of RNA and free NTPs).

To calculate the concentration of RNA, 5 μl of each elution can be mixedwith 995 μl of H₂O before measuring the OD of the mixture.

${n\; {mol}\mspace{14mu} {of}\mspace{14mu} {RNA}} = \frac{{OD} \times 40 \times 800\mspace{14mu} {\mu l} \times 1000}{330 \times 400 \times 5\mspace{11mu} {\mu l}}$

An alternative RNA purification procedure (LiCl precipitation) isexemplified below:

500 μL 10 M LiCl can be added to every 500 μl transcription reactionbefore freezing the mixture at −20° C. for 30 minutes to 1 hour. Themixture can then be applied for centrifugation at maximum speed for 20minutes. The resulting supernatant will be discarded, while the pelletcan be resuspended in 50 μl of 3M NaOAc and 50 μL of dH2O. A standardEtOH precipitation can be performed. The resulting pellet can bedissolved in 100 μL of dH2O and the RNA concentration can then bemeasured using Qubit.

Photoctrosslinking

Two types of Psoralen linkers are used: a 2′O-Me RNA linker PBI and aDNA linker DDB-1 that contain a biotin moiety to be used forstreptavidin assembly.

XB-PBI (SA/DNA display linker OMe RNA/DNA): (SEQ ID NO: 63)5′-(Psoralen C6) u agc gga (Biotin-dT)gc uaa ggA CGA-3′XB-DDB-1 (DNA display linker): (SEQ ID NO: 64)5′-(Psoralen C6) (C7-NH2-EZ biotin) T AGC GGA TGC TAA GGA CGA-3′

Psoralen C6

u,a,g,c=2-MeO-RNA

A,C,G=standard deoxy amidites

C7-NH2-amino spacer 7

EZ-biotin, Pierce EZ-link TFP-spacer-biotin

Reverse Frame 1 (translation of the linker regionas appears in a coding strand): (SEQ ID NO: 65) S S L A S A**Linker is light-sensitive and needs to be protected from light withaluminum foil etc.)

In the reaction set-up, the ratio of Linker/RNA can be from 1.5:1(maximum) to 1.1:1 (sufficient).

In Round 1 reaction, a large scale production is suggested (1-2 nmol ofRNA) to cover enough diversity. In later rounds, RNA input may bereduced to 100-600 pmol.

RNA 1 nmol linker (1 mM) 1.1 μL 10 X chemical ligation buffer 10 μLadding dH₂O to 100 μL (10 pmol RNA/μL final)

(The final concentration of RNA in ligation reaction works from 3-15pmol/ul. Ligation reaction volume can be varied.)

For annealing in a PCR thermocycler, the sample can be incubated at 85°C. for 30 seconds and to 4° C. at the rate of 0.3° C./second.

For irradiating, 1 μL of 100 mM DTT can be added to the annealedligation mix. The mix can be transferred to a thin wall 0.5 ml eppendorftube or kept in the same PCR tube. The irradiating process can be donewith a handheld multiwavelength UV Lamp (Uvp.com (Upland, Calif.)#UVGL-25) on ‘long wave’ (365 nm) for 6 minutes at room temperature.About 50-90% RNA will be expected to be ligated with the requirement ofpurification.

For QC of ligated RNA, a sample can be applied for electrophoresis on a6% TBU gel to check the ligation efficiency. Specifically, the 6% TBUgel can be pre-run under 300V for 15 minutes, followed by removing thecomb and flushing the wells thoroughly. Then for each well about 10-15pmol of free or ligated RNA can be mixed with 2× loading buffer (fromtranscription kit) and heated together at 90° C. for 3 minutes, followedby chilling on ice. Both pre-heated free and ligated RNA can then beloaded in wells of pre-heated gel for electrophoresis at 300V until theblue dye reaches the bottom.

An exemplary experiment was carried out as in FIG. 30. Specifically, KDRmRNA was in vitro transcribed and purified from the reaction by LiClprecipitation. One equivalent of PBI linker was annealed to mRNA sampleby heating to 85° C. followed by slow cooling to 4° C. in 1× X-linkbuffer. DTT was added to final concentration of 1 mM. The samples wereincubated at 365 nm wavelength for different amounts of time and thenresolved on a 6% TBU denaturing gel. The image is UV shadowing of thegel. As shown in FIG. 30, the crosslinking is observed after 5 minirradiation (Lane 7). Further a 6 min irradiation was used forphotocrosslinking.

Loading of Streptavidin

Streptavidin is a remarkably stable tetrameric protein which binds itsligand biotin at an exceptionally high affinity, Kd of 10⁻¹⁵ M.

A. With PBI Linkers

Due to high Tm of 0-Me RNA portion of PBI linker to RNA, it isrecommended to dilute the original 100 mM NaCl buffer (as in 1× X-linkbuffer) to 10-20 mM salt, which is 5-10× dilution with water. ½ to ¼equivalent of streptavidine (Prozyme, San Leandro, Calif.) solution canbe added in either PBS or 1× X-link buffer, e.g., 1-2 μM finalconcentration of streptavidine for 4 μM X-linked RNA. Then 1 μL ofRNAsine can be added and the mixture can be incubated at 48° C. in aheat block for 1 hour.

B. With DDB Linkers

Since a DDB linker is an all-DNA molecule, no dilution of the 1× X-linkbuffer (100 mM NaCl) is required. The DDB linker can accommodate betterSA loading, and it is possible to use only 1.5-2× excess of thecrosslinked mRNA to streptavidine. ½ or 1:1.5 equivalent ofstreptavidine (Prozyme, San Leandro, Calif.) solution can be added ineither PBS or 1× X-link buffer, e.g., 2-2.5 μM final concentration ofstreptavidine for 4 μM X-linked RNA. Then 1 μL of RNAsine can be addedand the mixture can be incubated at 48° C. in a heat block for 1 hour.

In each case (A or B) at least over 50% and up to 80% streptavidine canbe expected to be loaded.

Loading of Puromycin Linker BPP

BPP: 5′ Biotin-BB Cy3-(spacer 18)4-CC PuBPP-8: 5′ Biotin-BB Cy3-(spacer 18)8-CC Pu

Cy3 dye

4 or 8 units of Spacer 18

Pu=Puromycin-CPG

A BPP linker contains puromycin, a peptide acceptor molecule at the 3′end (the peptide acceptor enters the A site of the ribosome andcovalently couple to the COOH terminus of the nascent polypeptidechain). The linker has biotin at 5′ end which binds to streptavidinemolecule loaded on each X-linked mRNA molecule. This peptide acceptorwill ultimately enable the non-covalent tight association of the mRNA(genotype) to the protein encoded by this mRNA (phenotype). Each versionof a BPP linker (with a shorter 4× spacer-18 or with a longer 8×spacer-18) contains a fluorescent Cy-3 dye moiety (Ex. 550, Em. 570 nm)for visualization. Other fluorescent dyes can be used instead of Cy-3,such as Fluoresceins, BODIPY, Cy-5, rhodamines etc.

Specifically, 1 equivalent of a corresponding BPP linker (relative tothe amount of streptavidine) can be added to a loaded streptavidine-mRNAassembly. The mixture can be incubated for 15 minutes at roomtemperature. Almost 100% of BPP linker to streptavidine can be expectedfor binding.

An exemplary experiment was carried out as in FIG. 31. Specifically, KDRmRNA was in vitro transcribed and purified from the reaction by LiClprecipitation. One equivalent of either PBI or DDB linker was annealedto mRNA sample by heating to 85° C. followed by slow cooling to 4° C. in1× X-link buffer, containing 100 mM NaCl. Sample of PBI-x-linked mRNAwas then diluted 5× to final NaCl concentration of 20 mM. Sample ofDDB-x-linked mRNA was used undiluted. For streptavidine loading, about ½equivalent of streptavidin was added to each sample, followed by 1 hourincubation at either room temperature or at 50 C. Then BPP-Cy3 linkerwas added to each sample in amount, equivalent to streptavidine. After15 min incubation at room temperature the resulting assemblies wereresolved on a 4-16% NativePage.

Optional Step: Purification of the SA Assembly on Oligo dT Column

Since mRNA contains a tail of 20 As, it can be purified on an oligo dTcolumn at this step. This purification is not absolutely necessary, butmay somewhat improve the fusion yield at the next step.

Assembly Purification

Equal volume of 2× oligo dT binding buffer (200 mM Tris, pH 8, 2 M NaCl,20 mM EDTA, 0.1% Triton) can be added to the prepared mRNA-SA assembly.Then oligo-dT cellulose can be added into the mixture (100 mgtreated/washed oligo dT cellulose (1 mL of slurry) is sufficient tocapture up to 1 nmol RNA input) before rocking at 4° C. for 30-60minutes. The mixture can be spilled in a bench top centrifuge at 1500rpm for 3 minutes at 4° C. The resulting supernatant will be discarded.The resulting pellet can be resuspended in 700 μL of oligo dT washbuffer (100 mM Tris pH 8, 1 M NaCl, No EDTA, 0.05% Triton x-100), loadedonto a drip/spin column (BioRad #732-6204, Hercules, Calif.) and thenspilled in a microfuge at 1000 rpm for 10 seconds. The column can bewashed with 700 μL oligo dT wash buffer and spilled at 1000 rpm for 10more seconds. The wash step can be repeated for 8 times (during eachwash, the centrifuging rpm and time may be increased if it is gettinghard to spin through the wash buffer, but do not exceed 1500 rpm). 5 μLof the last wash can be collected for counting. Then the mRNA-SAassembly can be eluted with dH₂O. 60 μL dH₂O can be used for E1,followed by spinning at 1500 rpm for 10 seconds (5 μL used forcounting). For E2, 500 μL dH₂O can be added to the column. Afterincubation for 5 min at room temp, E2 can be collected after spinning at4000 rpm for 20 seconds (5 μL used for counting). For E3, 300 μL dH₂Ocan be added to and incubated with the column for 5 minutes at roomtemp. E3 can then be collected after spinning at 4000 rpm for 20 seconds(5 μL used for counting). In this Example, E2 contains 80% of themRNA-SA assembly.

Translation

The translation volume can be varied based on the amount of RNA input.An exemplary condition is given below:

Translation Set-Up (300 μL)

RNA-SA assembly 120 pmol + H₂O  82 μl (or to 300 μL) Amino acid mastermix (-met)  15 μl 10 mM Methionine  3 μl Lysate 200 μl Total volume 300μl

The mixture can be incubated at 30° C. in water bath or an incubator (inwater block) for 45 minutes to 1 hour.

Fusion Formation

100 μL 2 M KCl (500 mM final) can be mixed with 20 μL 1M MgCl₂ (50 mMfinal) and incubated at room temperature for 1 hour. The resultingmixture can be optionally frozen and stored at −20° C. for up to severaldays. Also, freezing somewhat improves fusion yield.

Then 50 μL 0.5M EDTA can be added to this mixture to disassemble theribosomes and produce ribosome-free fusions (10 μL saved for QC).

As an exemplary translation scale for selection, 1.2 nmol RNA (10×300μL) for Round 1, 600 pmol RNA (5×300 μL) for Round 2 and 120-600 pmolRNA (1-5×300 μL) for Round 3 and on.

Fusion Purification by Oligo dT Cellulose: PBI Linker

Oligo dT purification allows purification of RNA fusion molecules (plusRNA) and removes free protein molecules generated by in vitrotranslation. This procedure and the following Reverse Transcription stepshould be used when PBI linker assembly is translated. In case ofDDB-linker assembly a different procedure is recommended.

Fusion Purification

Equal volume of 2× oligo dT binding buffer (200 mM Tris, pH 8, 2 M NaCl,20 mM EDTA, 0.1% Triton) can be added to translation/fusion mix. Theoligo-dT cellulose (100 mg treated/washed oligo dT cellulose (1 mL ofslurry) is sufficient to capture translation/fusion up to 1 nmol RNAinput) can then be added to the mixture before rocking at 4° C. for30-60 minutes. After spinning in a bench top centrifuge at 1500 rpm for3 minutes at 4° C., the resulting supernatant will be discarded. Theresulting pellet can be resuspended in 700 μL oligo dT wash buffer (100mM Tris pH 8, 1 M NaCl, No EDTA, 0.05% Triton x-100) and loaded onto adrip/spin column (BioRad #732-6204, Hercules, Calif.), followed byspinning in a microfuge at 1000 rpm for 10 seconds. The resulting pelletcan be resuspended in 700 μL oligo dT wash buffer before spinning at1000 rpm for 10 seconds to reprecipitate the pellet. This wash step canbe repeated for 8 times (during each wash, the centrifuging rpm and timemay be increased if it is getting hard to spin through the wash buffer,but do not exceed 1500 rpm). 5 μL of the last wash can be collected forcounting. After washing, dH₂O can be used for elution. For example, 60μL dH₂O can be used for E1, followed by spinning at 1500 rpm for 10seconds (5 μL used for counting). For E2, 500 μL dH₂O can be added tothe column. After incubation for 5 min at room temp, E2 can be collectedafter spinning at 4000 rpm for 20 seconds (5 μL used for counting). ForE3, 300 μL dH₂O can be added to and incubated with the column for 5minutes at room temp. E3 can then be collected after spinning at 4000rpm for 20 seconds (5 μL used for counting). In this Example, E2contains 80% of the mRNA-SA assembly.

Estimation of Fusion Production:

The elution of fusions from the oligo dT cellulose can be furtheranalyzed by methods known in the art, e.g., gel-electrophoresis,fluorescence densitometry, or radiolabel counting, etc.

Reverse Transcription: PBI Linker

RNA-cDNA hybrid is made to stabilize and reduce the secondary structureof the RNA and to serve as a template for PCR after selection. In thecase of PBI linker, reverse transcription is preferred from an externalprimer (S6), but at a lower efficiency can be done from PBI linkeritself, since four 3′ nucleotides of the linker are DNA. This canpotentially convert the assembly into DNA-displayed fusions, though weuse a different all-DNA linker, DDB, for that purpose.

External RT primer: XB-S6-1: (SEQ ID No: 56)5'-TTAAAT AGC GGA TGC TAA GGA CGACTTGTCGTCGTCGTCCTTGTAGTC GGTTGGGGCGGATGCACTCCC-3' Reverse Frame 1:(SEQ ID No: 57) G S A S A P T D Y K D D D D K S S L A S A I (STOP)

Reaction Set-Up: (Invitrogen Reverse Transcription Kit)

The oligo-dT elutions can be spilled at 10000 rpm for 30 seconds toremove residual cellulose.

E2 + E3 supernatant from spin: 800 μl  (<1000 pmol total) (10-20 μL E2collected for electro- phoresis on gel) RT primer (S 6-1) 1 mM  1 μl(1000 pmol) 5 x first strand buffer 220 μl  0.1M DTT 11 μl (final 1 mM)25 mM dNTPs 22 μl (0.5 mM final each) Superscript II 25 μl H₂O 21 μlTotal 1100 μl  Incubate at 37° C. for 45-60 min (remove 10 μL to run ongel).

QC: cDNA synthesis can be monitored by gel electrophoresis usingNativePage and detected by fluorescence of the Cy-3 dye.

Fusion Purification by Oligo dT Cellulose, Coupled with the ReverseTranscription and RNAseH Digest: DDB Linker

By performing Oligo dT purification and reverse transcription on thecolumn in the mRNA-SA assembly with DDB linker we generate aDNA-displayed fusions. DDB in this case serves as an RT primer. The sameprocedure could be applied to PBI linker, which is also designed toserve as an internal RT primer, but demonstrated a lower efficiencycompared to DDB.

DNA-displayed fusions demonstrate a great stability to nuclease digestand are used for cell-based and in vivo selections.

Fusion Immobilization on the Oligo dT Cellulose

The equal volume of 2× oligo dT binding buffer (200 mM Tris, pH 8, 2 MNaCl, 20 mM EDTA, 0.1% Triton) can be added to translation/fusion mix.The oligo-dT cellulose (100 mg treated/washed oligo dT cellulose (1 mLof slurry) is sufficient to capture translation/fusion up to 1 nmol RNAinput) can then be further added before rocking at 4° C. for 30-60minutes. After spinning in a bench top centrifuge at 1500 rpm for 3minutes at 4° C., the resulting supernatant will be discarded. Theresulting pellet can be resuspended in 700 μL oligo dT wash buffer (100mM Tris pH 8, 1 M NaCl, No EDTA, 0.05% Triton x-100) and loaded onto adrip/spin column (BioRad #732-6204, Hercules, Calif.), followed byspinning in a microfuge at 1000 rpm for 10 seconds. The column is washedwith 700 μL oligo dT wash buffer, followed by spinning at 1000 rpm for10 seconds. The resulting pellet can be washed repeatedly for 8 times(during each wash, the centrifuging rpm and time may be increased if itis getting hard to spin through the wash buffer, but do not exceed 1500rpm). 5 μL of the last wash will be collected for counting.

Reverse Transcription on the Oligo dT Cellulose

The oligo dT resin with immobilized fusions can be equilibrated in 1×first strand buffer (prepare separately). The buffer contains 75 mM KCland 3 mM MgCl₂, which prevents elution of the fusions from the resin.

A mix can be prepared for the reverse transcription as following andthen incubated at 37-39° C. for 60-75 minutes.

Water 695 μl 5 x first strand buffer 200 μl 0.1M DTT 10 μl (1 mM final)25 mM dNTPs 40 μl (1 mM final each) Superscript II 50 μl RNasin 5 μlTotal 1000 μl

(In this example there is no external RT primer added. There isincreased amount of dNTPs and SSII reverse transcriptase.)

At this step the strand of cDNA is built which is covalently bound tothe biotin moiety and in turn tightly non-covalently attached to thestreptavidine-BPP-fusion sandwich, thus forming a precursor toDNA-displayed fusions.

RNAse H Digest of the RNA Strand and Elution from the Oligo dT Cellulose

Following the reverse transcription step, to the same column acorresponding amount of RNAse H can be added (10 μL of RNAse H (2 U/μL,Invitrogen, Carlsbad, Calif.)) for a 1000 μL reaction described above).Incubate for an additional 1 hour at 37° C.

The single-stranded DNA-fusions can be eluted by spinning down the oligodT column at 2000 rpm for 1 minute and then washed with 500 μL of 1×first strand buffer. In this Example around 80-95% of the material canbe eluted from the column.

Exemplary experiments were carried out as in FIGS. 32 and 33. As shownin FIG. 32, DNA display assembly (mRNA-xlinked with DDB linker) wastranslated in RRL for 1 hour at 30° C. at mRNA concentration of 200 nM.The product was loaded on oligo-dT column (Lane 2-3). The column waswashed several times with 1× oligo-dT buffer and then two times with 1×RT buffer, followed by dNTPs addition and SSII RT for 1 hour at 37° C.RNaseH treatment was further carried out for another 1 hour at 37° C.The column was eluted by spinning down (spin filter). In Lanes 4 and 5,mRNA assemblies with PBI linker were translated similarly to the above,following by treatment with oligo dT resin and elution with 5 mM Tris pH7.0.

In FIG. 33, DNA display assembly (mRNA-xlinked with DDB linker, lanes 1,2, and 3, or BPP linker, Lanes 4 and 5 (BPP-8 linker)) was translated inRRL for 1 hour at 30° C. at mRNA concentration of 200 nM. The productwas loaded on oligo-dT column. The column was washed several times with1× oligo-dT buffer and then 2× with 1× RT buffer, followed by dNTPsaddition and SSII RT for 1 hour at 37° C. RNaseH treatment was furthercarried out for another 1 hour at 37° C. The column was eluted byspinning down (spin filter). The fusions then were purified on an antiFlag M2 agarose (Lanes 2, 3 and 5).

2^(nd) strand synthesis and completion of the DNA-display fusion assembly. XB_S7 (T7TMVUTR2 42-mer; 5′PCR primer for both VH IgM, IgG libraries): (SEQ ID No: 52)5′-TAATACGACTCACTATAGGGACAATTACTATTTACAATTACA-3′XB_S7a (T7TMV primer 45 mer with additional ATG, improves Tm by 3 C):(SEQ ID No: 58) 5′-TAATACGACTCACTATAGGGACAATTACTATTTACAATTACAATG-3′

To the elution from the previous step, extra dNTPs and SSII RT can beadded, together with 1-1.5 equivalent of the TMV-T7 primer S7 or S7a.

An exemplary reaction includes mixing additional reagents per 1500 μLreaction, as described above:

S7 or S7a 1-1.5 equvalent SSII RT 10 μL dNTP 10 μL

The mixture can be incubated for an additional 1 hour at 37-39° C. Afterthe synthesis of the 2^(nd) strand the DNA displayed fusion assembly iscomplete and following additional purification on an anti FLAG antibodyresin, the fusions can be used for cell-based or in vivo selections.

An exemplary experiment is shown as in FIG. 34. The mRNA assembly wascarried out as following: 4 μM RNA and 4 μM linker (DDB or PBI) wereannealed 85 to 4° C. in 1× X-link buffer; then DTT was added to 1 mM andUV 365 for 6 min. Then SA was loaded as 1:2 ratio (final mRNA at 2 μMand SA at 1 μM) in 0.67× X-link buffer at 48° C. for 1 hour. PuromycinCy3 linker (BPP—4×C18 spacer) was added at 1 μM to the assembly for 10minutes at RT (room temperature). No oligo dT purification wasperformed. The following RT reaction was carried out as following:standard RT using superscript II (SSII) at 42° C. using 3 conditions:

DDB as RT primer, PBI as RT primer and S6 (external primer) in PBIassembly only. Each of the RT reactions were treated by RNAse H (2 U)for 1 hour at 37° C. Further, the second strand was synthesized byadding Y7 primer (T7-TMV) 1:1 and extra SSII RT enzyme and extra dNTPsfor 1 hour at 42° C. (conditions taken from Kurtz, Chembiochem 2001).

FLAG-Tag Purification

Flag purification recovers only full length protein molecules andremoves any free RNA and truncated protein molecules generated by invitro translation.

Binding and Wash:

500 μL M2 agarose (10 mM Gly pH 3.5 pre-stripped and pre-blocked in 1×binding buffer) suspension can be transferred to a 2 ml tube. Afterspinning in a microfuge at 1500 rpm for 1 minute, the resultingsupernatant will be discarded. The agarose can be further washed twicewith 1 mL flag binding buffer. The washed M2-agarose can then be mixedtogether with prepared chemically modified library (5 μl used forcounting) and ¼ library volume of 5× flag binding buffer and rocked at4° C. for 1 hour to overnight. After spinning in a microfuge at 1500 rpmfor 1 minute, the resulting supernatant will be transferred to a freshtube, while the resulting M2 agarose pellet will be resuspend in 0.7 mLflag binding buffer and loaded onto a drip spin column. The loadedcolumn can be spinned in a microfuge at 1000 rpm for 10 seconds andfurther washed 6 times with 0.7 ml flag binding buffer, followed byspinning at 1000 rpm for 10 seconds. Then the column can be washed twicewith 0.7 ml 1× binding buffer, followed by spinning at 1000 rpm for 10seconds. (5 μL of last wash can be collected for counting).

Elution:

500 μL of 100 μg/mL FLAG peptide in 1× binding buffer can be added tothe column. After incubating for 5 minutes, the column can be spinningat 3000 rpm before collecting the elution. The elution process can berepeated with 300 μl flag peptide (5 μL of each elution can be collectedfor counting).

Estimation of Recovery:

Routinely the recovery rate can be around 10-30%. The recovery can bemeasured by fluorescence or other methods known in the art.

PCR

Depending on the purpose, Taq polymerase or high fidelity polymerase canbe used for amplification. Small scale PCR is recommended to check outthe PCR cycles to prevent PCR artifacts usually caused by overamplification.

Reaction Set-Up:

10X Herculase Buffer (Strategene) 2.5 μl 10 mM dNTP 0.5 μl T7 TMV UTR (5μM) 1 μl Cμ2flagA20 (5 μM) 1 μl Template 2.5 μl H₂O 17 μl Herculase(Stratagene) 0.5 μl 25 μl

Note: The fractions collected in previous steps, e.g., flow-through,last wash, Elution 1, and Elution 2, can be used as template for PCR.Specifically, 2.5-5 μL of template can be used in this PCR reaction,especially for early selection rounds (round 1-3, for example). However,it may be helpful to use less. An exemplary PCR condition is listedbelow:

95° C. for 3 minutes 1 cycle 95° C. for 30 seconds 50° C. for 30 secondsX cycles (X = 15, 20, or 25 72° C. for 1 minute {close oversize brace}depending on the signal) 72° C. for 5 minutes 1 cycle

Note on Tm: Tm is calculated to the primer. In this Example, primers canhave Tms higher than 50° C. or 52 to 65° C.

After the PCR reaction, 5 μL of reaction mixture can be used forelectrophoresis on a 2% E-gel. A major band having a size of −400 bp canbe expected on the gel. If the quality of small-scale PCR is acceptable,the rest of elution can be used as template for large-scale PCR underthe similar conditions.

DNA Purification:

Sometimes it may be necessary to purify the PCR product. Afterelectrophoresis, the gel slice on 2% agarose gel containing the PCRproduct (the 400 bp band) can be cut out under UV light. DNA can then beextracted from the gel slice using Qiaquick Gel Extraction Kit (Qiagen#28706, Valencia, Calif.). 5 μL of purified DNA can be used forelectrophoresis on a 2% E-gel. Usually ˜80% recovery can be expected.

Tagging for 454 Sequencing: DNA Display

For the tagging of individual molecules at non-amplification selectionstep the following 2^(nd) strand primer is used:

(SEQ ID No: 59) GCCTCCCTCGCGCCATCAGNNNNNNGGGACAATTACTATTTACAATTAC A ATG

This primer contains TMV sequence, a 454 adaptor sequence and N6 randomtag. The corresponding PCR primer for the 3′ end (contains a second 454adaptor, Tm of gene-specific part is 50.4 C)

(SEQ ID No: 60) GCCTTGCCAGCCCGCTCAGTAGCGGATGCTAAGCACGA

Amplicon preparation is performed using adaptor primers only:

Forward: Tm 64.7° C. GCCTCCCTCGCGCCATCAG (SEQ ID No: 61)Reverse: Tm 65.7° C. GCCTTGCCAGCCCGCTCAG (SEQ ID No: 62)

Tm of both are around 65° C., thus RACE type of PCR is possible for a 3′primer.

EQUIVALENTS

Numerous modifications and alternative embodiments of the presentinvention will be apparent to those skilled in the art in view of theforegoing description. Accordingly, this description is to be construedas illustrative only and is for the purpose of teaching those skilled inthe art the best mode for carrying out the present invention. Details ofthe structure may vary substantially without departing from the spiritof the invention, and exclusive use of all modifications that comewithin the scope of the appended claims is reserved. It is intended thatthe present invention be limited only to the extent required by theappended claims and the applicable rules of law.

All literature and similar material cited in this application,including, patents, patent applications, articles, books, treatises,dissertations, web pages, figures and/or appendices, regardless of theformat of such literature and similar materials, are expresslyincorporated by reference in their entirety. In the event that one ormore of the incorporated literature and similar materials differs fromor contradicts this application, including defined terms, term usage,described techniques, or the like, this application controls. Suchequivalents are intended to be encompassed by the following claims.

1. An X-display complex comprising: (a) a first nucleic acid moleculecomprising a polypeptide-encoding sequence; (b) a polypeptide encoded bythe first nucleic acid molecule; and (c) a second nucleic acid moleculecomprising a nucleic sequence complementary to a portion of the firstnucleic acid molecule, wherein the first nucleic acid molecule is boundto the second nucleic molecule through complementary nucleic acid basepairing, and wherein the second nucleic acid molecule is non-covalentlybound to the polypeptide.
 2. The complex of claim 1, further comprising:(a) a high affinity ligand covalently bound to the second nucleic acidmolecule; and (b) a ligand acceptor bound to a peptide acceptor, whereinthe high affinity ligand is bound to the ligand acceptor and the peptideacceptor is covalently bound to the polypeptide.
 3. The complex of claim2, wherein one or more of the following conditions are met: (a) thecomplex further comprises a second high affinity ligand, wherein thesecond high affinity ligand is covalently bound to the peptide acceptor,and wherein the second high affinity ligand is bound to the ligandacceptor; and (b) the ligand acceptor is covalently bound to the peptideacceptor.
 4. The complex of claim 3, wherein one or more of thefollowing conditions are met: (a) the peptide acceptor is bound to thesecond high affinity ligand through a linker; (b) the linker comprisespolyethylene glycol; and (c) the linker comprises a polysialic acidlinker. 5-7. (canceled)
 8. The complex of claim 1, wherein one or moreof the following conditions are met: (a) the complex further comprises:(i) a ligand acceptor covalently bound to the second nucleic acidmolecule; and, (ii) a high affinity ligand bound to a peptide acceptor,wherein the ligand acceptor is bound to the high affinity ligand and thepeptide acceptor is covalently bound to the C-terminus of thepolypeptide; (b) the complex further comprises a third nucleic acidmolecule, comprising a nucleic sequence complementary to a portion ofthe second nucleic acid molecule, wherein the third nucleic acidmolecule is bound to the second nucleic molecule through complementarynucleic acid base pairing, wherein the third nucleic acid molecule iscovalently bound to a peptide acceptor, and wherein the peptide acceptoris covalently bound to the polypeptide (c) the second nucleic acidmolecule is a branched nucleic acid molecule; and (d) the second nucleicacid molecule is capable of acting as a primer for reverse transcriptionof the first nucleic acid molecule. 9-11. (canceled)
 12. An X-displaycomplex comprising: (a) a nucleic acid molecule comprising apolypeptide-encoding sequence, covalently bound to a first high affinityligand; (b) a polypeptide encoded by the nucleic acid molecule; (c) aligand acceptor bound to a peptide acceptor, wherein the high affinityligand is bound to the ligand acceptor and the peptide acceptor iscovalently bound to the C-terminus of the polypeptide.
 13. The complexof claim 12, wherein one or more of the following conditions are met: a)the ligand acceptor is covalently bound to the peptide acceptor; and b)the complex further comprises a second high affinity ligand, wherein thesecond high affinity ligand is covalently bound to the peptide acceptor,and wherein the second high affinity ligand is bound to the ligandacceptor.
 14. (canceled)
 15. An X-display complex comprising: (a) anucleic acid molecule comprising a polypeptide-encoding sequence,covalently bound to a first high affinity ligand; (b) a first ligandacceptor covalently bound to a second high affinity ligand; and, (c) apolypeptide encoded by the first nucleic acid molecule, wherein thepolypeptide comprises a second ligand acceptor, wherein the first highaffinity ligand is bound to the first ligand acceptor, and the secondhigh affinity ligand is bound to the second ligand acceptor.
 16. Thecomplex of claim 15, wherein one or more of the following conditions aremet: (a) the first high affinity ligand or second high affinity ligandis biotin; (b) the first high affinity ligand or second high affinityligand is selected from the group comprising FK506, methotrexate,PPI-2458, biotin, hirudin, ZFVp(O)F, fluorescein-biotin, ABD (albuminbinding domain), 18 bp DNA, RNAse A, cloroalkanes, aryl (beta-aminoethyl) ketones, and protein A; (c) the first ligand acceptor or secondligand acceptor is selected from the group comprising FKBP12,dihydrofolate reductase, methionine aminopeptidase, dimericstreptavidin, streptavidin tetramer, thrombin, carboxypeptidase,monovalent Ab, HSA (albumin), Zn finger, hRI (RNase inhibitor), mutatedhaloalkane dehalogenase, haloTag, and sortase; (d) the first ligandacceptor or second ligand acceptor is streptavidin and (e) thepolypeptide is chosen from the group comprising an antibody, a VHdomain, a VL domain, a Fab fragment, a single chain antibody, ananobody, a unibody, an adnectin, an affibody, a DARPin, an anticalin,an avimer, a 10Fn3 domain, and a versabody. 17-20. (canceled)
 21. AnX-display complex comprising: (a) a nucleic acid molecule comprising apolypeptide-encoding sequence, covalently attached to a high affinityligand; and (b) a polypeptide encoded by the first nucleic acidmolecule, wherein the polypeptide comprises a ligand acceptor, whereinthe ligand is bound to the ligand acceptor.
 22. The complex of claim 21,wherein the ligand is FK506 and the ligand acceptor is the FK506-bindingdomain of FKBP.
 23. The complex of claim 1, wherein one or more of thefollowing conditions are met: (a) the first nucleic acid molecule isselected from the group consisting of ssRNA, ssDNA, ssDNA/RNA hybriddsDNA, and dsDNA/RNA hybrid; (b) the polypeptide-encoding sequence ofthe first nucleic acid molecule does not contain an in-frame stop codon;(c) the polypeptide is a binding protein; (d) the binding protein is theVH or VL domain of an antibody; and (e) the complex does not contain aribosome. 24-27. (canceled)
 28. A library comprising a plurality of theX-display complexes of claim 1, wherein at least a portion of thecomplexes contain different polypeptide-encoding sequences.
 29. A methodof producing a library of X-display complexes comprising the steps of:(a) providing a library of mRNA sequences comprising a sequence elementcomplementary to a first nucleic acid linker (b) providing a firstnucleic acid linker operably linked to a first high affinity ligand suchthat the first nucleic acid linker binds to the mRNA throughcomplementary nucleic acid base pairing (c) providing second highaffinity ligand operably linked to a peptide acceptor (d) providing aligand acceptor with at least two binding sites or providing at leastsuch that the ligand acceptor binds to the first high affinity ligandand the second high affinity ligand (e) allowing translation of the mRNAto occur such that the peptide acceptor binds to the translated proteinthereby forming a nucleic acid-polypeptide complex linking the mRNA tothe protein.
 30. The method of claim 29, further comprising allowingreverse transcription of the mRNA using the first nucleic acid linker inthe nucleic acid-polypeptide complex as a primer such that a DNA/RNAhybrid is formed.
 31. The method of claim 30, further comprisingdegrading the mRNA and synthesizing a complementary DNA strand therebyforming a DNA/DNA hybrid in the nucleic acid-polypeptide complex. 32.The method of claim 29, wherein one or more of the following conditionsare met: (a) the mRNA further comprises a TMV enhancer; (b) the mRNAfurther comprises a Cμ sequence; (c) the mRNA further comprises a FLAGtag; (d) the mRNA further comprises an SA display sequence; (e) the mRNAfurther comprises as A20 tail. 33-36. (canceled)
 37. A library ofnucleic acid-polypeptide complexes produced by the method of claim 29.38. A method of selecting an isolated nucleic acid molecule encoding apolypeptide capable of binding to an antigen of interest, comprising thesteps of: (a) providing the library of nucleic acid-polypeptidecomplexes of claim 28; (b) contacting the library with an antigen ofinterest; (c) selecting from the library at least one nucleicacid-polypeptide complex that binds to the antigen of interest; and, (d)isolating the polypeptide-encoding sequence of the selected nucleicacid-polypeptide complex.
 39. A method of producing a polypeptidecapable of binding to an antigen of interest, comprising: (a) providinga polypeptide-encoding sequence selected using the method of claim 38;and (b) expressing the polypeptide encoded by the polypeptide-encodingsequence.
 40. An isolated nucleic acid molecule encoding a polypeptidecapable of binding to an antigen of interest, selected by the method ofclaim
 29. 41. An X-display complex comprising: (a) a first nucleic acidmolecule comprising a polypeptide-encoding sequence; (b) a polypeptideencoded by the first nucleic acid molecule; and (c) a second nucleicacid molecule comprising a nucleic sequence complementary to a portionof the first nucleic acid molecule, (d) a first high affinity ligandcovalently bound to the second nucleic acid molecule, (e) a first ligandacceptor, (f) a second high affinity ligand covalently bound through viaone or more linking molecules to a peptide acceptor, wherein the firstnucleic acid molecule is bound to the second nucleic molecule throughcomplementary nucleic acid base pairing, wherein the first high affinityligand is noncovalently bound to the ligand acceptor at a first bindingsite, wherein the second ligand is noncovalently bound to the ligandacceptor at a second binding site, and wherein the one or more linkingmolecules are polyethylene glycol molecules.
 42. The complex of claim 41wherein one or more of the following conditions are met: (a) the firsthigh affinity ligand is biotin and the ligand acceptor is selected fromthe group comprising streptavidin, dimeric streptavidin, and tetramericstreptavidin; (b) the second high affinity ligand is biotin and theligand acceptor is selected from the group comprising streptavidin,dimeric streptavidin, and tetrameric streptavidin; (c) the first highaffinity ligand or second high affinity ligand is selected from thegroup comprising FK506, methotrexate, PPI-2458, biotin, hirudin,ZFVp(O)F, fluorescein-biotin, ABD (albumin binding domain), 18 bp DNA,RNAse A, cloroalkanes, aryl (beta-amino ethyl) ketones, and protein A;(d) the complex further comprises a second ligand acceptor; (e) thepeptide acceptor is puromycin; (f) the first or second nucleic acidmolecule further comprises psoralen; and (g) the polypeptide is chosenfrom the group comprising an antibody, a VH domain, a VL domain, a Fabfragment, a single chain antibody, a nanobody, a unibody, an adnectin,an affibody, a DARPin, an anticalin, an avimer, a ¹⁰Fn3 domain, and aversabody. 43-45. (canceled)
 46. The complex of claim 42 wherein thesecond ligand acceptor is selected from the group comprising FKBP12,dihydrofolate reductase, methionine aminopeptidase, dimericstreptavidin, Streptavidin tetramer, thrombin, carboxypeptidase,monovalent Ab, HSA (albumin), Zn finger, hRI (RNase inhibitor), mutatedhaloalkane dehalogenase, haloTag, and sortase. 47-49. (canceled)
 50. Aheterobifunctional complex comprising: (a) a first high affinity ligandcovalently bound to a nucleic acid molecule; (b) a second high affinityligand covalently bound to a peptide acceptor; and (c) a ligand acceptorcomprising two or more ligand binding sites; wherein the first andsecond are bound to the ligand acceptor at distinct ligand bindingsites.
 51. The complex of claim 50 wherein one or more or the followingconditions are met: (a) the first high affinity ligand and second highaffinity ligand are identical; (b) the first high affinity ligand andsecond high affinity ligand are biotin; (c) the nucleic acid moleculecomprises a psoralen moiety; (d) the peptide acceptor is puromycin; (e)the ligand acceptor is a multimeric protein; and (f) the ligand acceptoris streptavidin. 52-56. (canceled)